TW202434286A - Compositions and methods of treating childhood onset idiopathic nephrotic syndrome - Google Patents

Abstract

The present disclosure provides methods for treating childhood-onset idiopathic nephrotic syndrome (INS), or reducing risk and/or frequency of relapse from childhood-onset INS, in an individual that is greater than or equal to 2 years of age and less than or equal to 25 years of age. In some embodiments, the methods comprise administering to the individual an effective amount of obinutuzumab.

Description

Compositions and methods for treating childhood-onset idiopathic nephrotic syndrome

Provided herein are methods of treating childhood-onset idiopathic nephrotic syndrome (INS) in an individual ( e.g. , an individual aged 2 years or older and 25 years or younger) or reducing the risk and/or frequency of recurrence of childhood-onset INS by administering a type II anti-CD20 antibody.

Childhood-onset idiopathic nephrotic syndrome (INS) is also called primary nephrotic syndrome (excluding secondary causes) and encompasses minimal change disease (MCD) and focal and segmental glomerulosclerosis (FSGS). Although rare, the incidence of childhood-onset INS varies by race and region and is higher in certain ethnic groups, specifically South Asians and African Americans, where genetic influences contribute to a higher risk, specifically for FSGS (Chanchlani and Parekh (2016) Front Pediatr 4:39). The disease is usually first diagnosed between the ages of 2 and 5 years (in 70% of patients with MCD), generally affects boys more than girls (2:1), and is defined by the presence of nephrotic-range proteinuria, edema, hyperlipidemia, and hypoalbuminemia (Noone et al. (2018) Lancet 392:61-74).

Patients with childhood-onset INS are initially treated with systemic oral corticosteroids. The frequency of relapses and the response to corticosteroid treatment allow the disease to be classified into subtypes that reflect the severity of the disease. These subtypes include "steroid-resistant nephrotic syndrome (potential genetic cause)", "non-/rare recurrent steroid-sensitive nephrotic syndrome", "FRNS", and "SDNS" (Noone et al. (2018) Lancet 392:61-74). In the latter two subtypes, patients receive multiple courses of steroids during episodes and often continue to receive maintenance steroid therapy to prevent further relapses. The optimal goal of treatment is to maximize steroid sparing while limiting recurrence rate based on the patient's clinical response and drug-related adverse effects.

Childhood-onset INS recurs in more than 75% of patients, and almost 50% of patients demonstrate frequent relapses or steroid dependence (Abdel-Hafez et al. (2017) J. Nephropathol 6:180-186). In children whose disease is refractory to steroids, various steroid immunosuppressants (e.g., cyclophosphamide, levamisole, cyclosporine A, tacrolimus, MMF, and rituximab) have been shown to reduce the risk of relapse. However, high-quality head-to-head data comparing these drugs are limited (Mason et al. (2020) Clin. J. Am. Soc. Nephrol. 15:983-994).

The anti-CD20 monoclonal antibody rituximab was first described in 2004 for the treatment of childhood-onset INS (Benz et al. (2004) Pediatr Nephrol 19(7):794-797) and has since emerged as a promising unapproved treatment option in both children and adults with FRNS or SDNS. Multiple investigator-initiated trials and case reports of its use have been described in the literature with varying clinical response rates (renal remission [freedom from relapse] in 50%-85% of patients at 12 months), and rituximab has been shown to reduce the annualized relapse rate in patients with FRNS and SDNS. However, patients who do not achieve complete remission at 12 months generally relapse between 8 and 9 months after rituximab treatment, and most of these relapses occur in the context of B cell recovery/reconstitution (Iijima et al. (2014) Lancet 384:1273-1281; Colucci et al. (2016) J. Am Soc Nephrol 27:1811-1822).

Delayed reconstitution of the memory B cell pool is associated with prolonged remission despite tapering or cessation of concomitant immunosuppression (Colucci et al. (2019) Front Immunol 10:1653). Obinutuzumab (GAZYVA®, GAZYVARO®) is a humanized, glycoengineered type II anti-CD20 antibody that has enhanced depletion of B cells in peripheral blood and tissues relative to type I antibodies such as rituximab and ofatumumab. It is administered as an IV infusion. Consistent with its more effective B-cell depletion, obinutuzumab is superior to rituximab in combination with standard chemotherapy for the treatment of adult chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL) and is currently approved worldwide for these indications. Obinutuzumab is also indicated for the treatment of patients with FL who have not responded to rituximab or a rituximab-containing regimen or whose disease has progressed during or after treatment. In addition, obinutuzumab is currently in clinical development for autoimmune diseases (lure nephritis [LN], membranous nephropathy, and systemic lupus erythematosus [SLE]) in adults and children.

Despite current treatments for childhood-onset INS, patients remain at significant risk for impaired growth and other significant side effects of steroid-related toxicity. Given the high rates of clinical relapse following immunosuppressive therapy and the associated potential risk of end-stage renal disease in childhood-onset INS, there remains an unmet need for approved steroid-sparing therapies with improved efficacy and better long-term outcomes.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

In certain aspects, provided herein is a method of treating childhood-onset INS in an individual, comprising administering to the individual a first antibody exposure to a Type II anti-CD20 antibody and a second antibody exposure to the Type II anti-CD20 antibody; wherein the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the first antibody exposure comprising: (a) a total exposure of between about 1800 mg and about 2200 mg of the Type II anti-CD20 antibody, or (b) a total exposure of between about 36 mg/kg and about 44 mg/kg of the Type II anti-CD20 antibody, provided that the individual weighs less than 45 kg; wherein the second antibody exposure comprises the Type II anti-CD20 antibody; The invention relates to a method of administering to an individual a total of one or two doses of a Type II anti-CD20 antibody, wherein the second antibody exposure comprises: (c) a total exposure of between about 1800 mg and about 2200 mg of a Type II anti-CD20 antibody, or (d) a total exposure of between about 36 mg/kg and about 44 mg/kg of a Type II anti-CD20 antibody, provided that the individual weighs less than 45 kg; wherein the Type II anti-CD20 antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO:1, the HVR-H2 sequence of SEQ ID NO:2, and the HVR-H3 sequence of SEQ ID NO:3, and a light chain comprising the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of SEQ ID NO:5, and the HVR-L3 sequence of SEQ ID NO:6. sequence; and wherein the individual is a human who is greater than or equal to 2 years old and less than or equal to 25 years old. Also provided herein is a method of preventing relapse, reducing the risk of relapse, and/or reducing the frequency of relapse in an individual with childhood-onset idiopathic nephrotic syndrome (INS), comprising administering to the individual a first antibody exposure to a Type II anti-CD20 antibody and a second antibody exposure to the Type II anti-CD20 antibody; wherein the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the first antibody exposure comprising: (a) a total exposure of between about 1800 mg and about 2200 mg of the Type II anti-CD20 antibody, or (b) between about 36 mg/kg and about 44 mg/kg of the Type II anti-CD20 antibody. wherein the second antibody exposure comprises one or two doses of a Type II anti-CD20 antibody, the second antibody exposure comprising: (c) a total exposure of between about 1800 mg and about 2200 mg of the Type II anti-CD20 antibody, or (d) a total exposure of between about 36 mg/kg and about 44 mg/kg of the Type II anti-CD20 antibody, provided that the individual weighs less than 45 kg; wherein the Type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprising the HVR-H1 sequence of SEQ ID NO:1, the HVR-H2 sequence of SEQ ID NO:2, and the HVR-H3 sequence of SEQ ID NO:3, and the light chain comprising the HVR-L1 sequence of SEQ ID NO:4 sequence, the HVR-L2 sequence of SEQ ID NO:5, and the HVR-L3 sequence of SEQ ID NO:6; and wherein the individual is a human who is greater than or equal to 2 years old and less than or equal to 25 years old.

In some embodiments, the individual weighs greater than or equal to 45 kg. In some embodiments, the first antibody exposure comprises a total exposure of between about 1800 mg and about 2200 mg of Type II anti-CD20 antibody; the second antibody exposure comprises a total exposure of between about 1800 mg and about 2200 mg of Type II anti-CD20 antibody; and the individual weighs greater than or equal to 45 kg.

In some embodiments, the first antibody exposure comprises a first dose of a Type II anti-CD20 antibody between about 900 mg and about 1100 mg, and a second dose of a Type II anti-CD20 antibody between about 900 mg and about 1100 mg. In some embodiments, the first antibody exposure comprises a first dose of a Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and a second dose of a Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and wherein the individual weighs less than 45 kg. In some embodiments, the first antibody exposure comprises a first dose of a Type II anti-CD20 antibody and a second dose of the Type II anti-CD20 antibody, and wherein the second dose of the first antibody exposure is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the first antibody exposure. In some embodiments, the first antibody exposure comprises a first dose of a Type II anti-CD20 antibody and a second dose of the Type II anti-CD20 antibody, and wherein the second dose of the first antibody exposure is not provided until about 2 weeks after the first dose of the first antibody exposure. In some embodiments, the first dose of the first antibody exposure is about 1000 mg of the Type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure is about 1000 mg of the Type II anti-CD20 antibody. In some embodiments, the first dose of the first antibody exposure is about 20 mg/kg of Type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. In some embodiments, the second dose of the first antibody exposure is about 20 mg/kg of Type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. In some embodiments ( e.g. , wherein the dose of the first antibody exposure is a fixed dose), the individual weighs greater than or equal to 45 kg.

In some embodiments, the second antibody exposure comprises a first dose of a Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and a second dose of a Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and wherein the individual weighs less than 45 kg. In some embodiments, the second antibody exposure comprises a first dose of a Type II anti-CD20 antibody and a second dose of the Type II anti-CD20 antibody, and wherein the second dose of the second antibody exposure is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the second antibody exposure. In some embodiments, the second dose of the second antibody exposure is not provided until about 2 weeks after the first dose of the second antibody exposure. In some embodiments, the first dose of the second antibody exposure is about 1000 mg of the Type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure is about 1000 mg of the Type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure is about 20 mg/kg of Type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. In some embodiments, the second dose of the second antibody exposure is about 20 mg/kg of Type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. In some embodiments ( e.g. , wherein the dose of the second antibody exposure is a fixed dose), the individual weighs greater than or equal to 45 kg.

In some embodiments, the first and/or second antibody exposure is administered intravenously.

In some embodiments, the individual has or has been diagnosed with childhood-onset INS. In some embodiments, the individual has Frequently Recurrent Nephrotic Syndrome (FRNS). In some embodiments, the childhood-onset INS is steroid-dependent nephrotic syndrome (SDNS). In some embodiments, for example, prior to administration of the first antibody exposure, the individual is in complete remission.

In some embodiments, the method further comprises administering an effective amount of a glucocorticoid or corticosteroid to the subject. In some embodiments, the glucocorticoid or corticosteroid comprises methylprednisolone. In some embodiments, methylprednisolone is administered to the subject in a dosage of about 80 mg. In some embodiments, for example, if the subject weighs less than 45 kg, methylprednisolone is administered intravenously to the subject in a dosage of 1.5 mg/kg. In some embodiments, the glucocorticoid or corticosteroid comprises prednisone. In some embodiments, the method further comprises administering an effective amount of an antihistamine to the subject. In some embodiments, the antihistamine comprises diphenhydramine. In some embodiments, diphenhydramine is administered orally at a dose of 0.5-1 mg/kg, optionally up to a maximum dose of 50 mg. In some embodiments, the method further comprises administering to the subject an effective amount of acetaminophen. In some embodiments, acetaminophen is administered orally at a dose of 15 mg/kg, optionally up to a maximum dose of 1000 mg.

In some embodiments, the method results in a sustained complete remission in the individual at 1 year. In some embodiments, the method produces a depletion of circulating peripheral B cells in the individual. In some embodiments, the circulating peripheral B cells are CD19+ B cells. In some embodiments, the B cells are naïve B cells ( e.g., CD19+ CD27- B cells), memory B cells ( e.g., CD19+ CD27+ B cells), or plasmablasts ( e.g., CD19+ CD27+ CD38++ B cells). In some embodiments, the B cells are CD19+CD3-CD14-CD33-CD56- cells. In some embodiments, after administration of the type II anti-CD20 antibody, B cells are depleted to a level where circulating peripheral B cells are present in the individual's peripheral blood at about 5 cells per µL or less. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present at about 1 cell/µL or less in the peripheral blood from the individual. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present at about 0.5 cells/µL or less in the peripheral blood from the individual. In some embodiments, B cell depletion to a level of circulating peripheral B cells present in the individual's peripheral blood is achieved after the first antibody exposure. In some embodiments, B cells are depleted to a level below the limit of detection using HSFC. In some embodiments, the HSFC has a lower limit of quantification (LLOQ) for B cells of about 1.0 cells per µL or less, about 0.8 cells per µL or less, about 0.6 cells per µL or less, about 0.5 cells per µL or less, or 0.441 cells per µL or less. In some embodiments, B cell depletion persists for at least 52 weeks after the first dose of the first antibody exposure. In some embodiments, after administration of the type II anti-CD20 antibody, the subject's circulating peripheral B cells are depleted by at least about 90% compared to corresponding measurements in the same subject before administration of the type II anti-CD20 antibody, or compared to corresponding measurements in a subject not receiving treatment with the type II anti-CD20 antibody.

In some embodiments, the first antibody exposure comprises two doses of 1000 mg of a Type II anti-CD20 antibody on days 1 and 15 of treatment; the second antibody exposure comprises two doses of 1000 mg of a Type II anti-CD20 antibody on days 168 and 182 of treatment; and the Type II anti-CD20 antibody is obinutuzumab. In some embodiments, the first antibody exposure comprises two doses of 20 mg/kg of a Type II anti-CD20 antibody on days 1 and 15 of treatment; the second antibody exposure comprises two doses of 20 mg/kg of a Type II anti-CD20 antibody on days 168 and 182 of treatment; the Type II anti-CD20 antibody is obinutuzumab, and the individual weighs less than 45 kg. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of a Type II anti-CD20 antibody at Week 0 and Week 2 of treatment; the second antibody exposure comprises two doses of 1000 mg of a Type II anti-CD20 antibody at Week 24 and Week 26 of treatment; and the Type II anti-CD20 antibody is obinutuzumab. In some embodiments, the first antibody exposure comprises two doses of 20 mg/kg of a Type II anti-CD20 antibody at Week 0 and Week 2 of treatment; the second antibody exposure comprises two doses of 20 mg/kg of a Type II anti-CD20 antibody at Week 24 and Week 26 of treatment; the Type II anti-CD20 antibody is atezolizumab; and the individual weighs less than 45 kg. In some embodiments ( e.g. , where the dose of antibody exposure is a fixed dose), the individual weighs greater than or equal to 45 kg.

In certain aspects, provided herein is a method for treating childhood-onset INS in a subject or reducing the risk and/or frequency of recurrence thereof, comprising administering to the subject intravenously a first and a second antibody exposure to a type II anti-CD20 antibody; wherein the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody at week 0 and week 2 of treatment; wherein the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody at week 24 and week 26 of treatment; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the The invention relates to a method for treating a human subject comprising: a HVR-H3 sequence of SEQ ID NO:3, the light chain comprising the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of SEQ ID NO:5, and the HVR-L3 sequence of SEQ ID NO:6; wherein the subject is a human aged 2 years or more and less than or equal to 25 years old; and wherein the subject weighs greater than or equal to 45 kg. In certain aspects, provided herein is a method for treating childhood-onset INS in a subject or reducing the risk and/or frequency of recurrence thereof, comprising administering to the subject intravenously a first and a second antibody exposure to a type II anti-CD20 antibody; wherein the first antibody exposure comprises two doses of 20 mg/kg of the type II anti-CD20 antibody at week 0 and week 2 of treatment; wherein the second antibody exposure comprises two doses of 20 mg/kg of the type II anti-CD20 antibody at week 24 and week 26 of treatment; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the The invention relates to a method for treating a human subject comprising: a HVR-H3 sequence of SEQ ID NO:3, the light chain comprising the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of SEQ ID NO:5, and the HVR-L3 sequence of SEQ ID NO:6; wherein the subject is a human aged 2 years or more and less than or equal to 25 years old; and wherein the subject weighs greater than or equal to 45 kg. In certain aspects, provided herein is a method for treating childhood-onset INS in an individual or reducing the risk and/or frequency of recurrence thereof, comprising administering to the individual intravenously a first and a second antibody exposure to a type II anti-CD20 antibody; wherein the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on day 1 and day 15 of treatment; wherein the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on day 168 and day 182 of treatment; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3. The invention relates to a method for treating a human subject of the present invention wherein the light chain comprises an HVR-L1 sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and an HVR-L3 sequence of SEQ ID NO:6; wherein the subject is a human aged 2 years or more and less than or equal to 25 years old; and wherein the subject weighs greater than or equal to 45 kg. In certain aspects, provided herein is a method for treating childhood-onset INS in a subject or reducing the risk and/or frequency of recurrence thereof, comprising administering to the subject intravenously a first and a second antibody exposure to a type II anti-CD20 antibody; wherein the first antibody exposure comprises two doses of 20 mg/kg of the type II anti-CD20 antibody on day 1 and day 15 of treatment; wherein the second antibody exposure comprises two doses of 20 mg/kg of the type II anti-CD20 antibody on day 168 and day 182 of treatment; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3. The invention relates to a method for treating a type II anti-CD20 antibody comprising: a HVR-H3 sequence, a light chain comprising an HVR-L1 sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and an HVR-L3 sequence of SEQ ID NO:6; wherein the subject is a human aged 2 years or more and less than or equal to 25 years old; and wherein the subject weighs greater than or equal to 45 kg. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

In some embodiments of the methods described herein, the type II anti-CD20 antibody is a humanized antibody. In some embodiments, the type II anti-CD20 antibody is afucosylated. In some embodiments, the heavy chain of the type II anti-CD20 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the light chain of the type II anti-CD20 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the type II anti-CD20 antibody comprises the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and the light chain variable region comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the type II anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

In some embodiments, the methods further comprise administering prednisone ( e.g. , orally) to the subject. In some embodiments, oral prednisone is administered to the subject at a dose of 0.5-1 mg/kg/day up to a maximum of 60 mg/day. In some embodiments, oral prednisone is administered to the subject at a dose of 0.5-1 mg/kg/day up to week 2, then tapered to a dose of 5 mg/day at week 24 of treatment. In some embodiments, oral prednisone is administered to the subject at a dose of 0.5-2 mg/kg/day up to a maximum of 60 mg/day. In some embodiments, oral prednisolone is administered to the subject at a dose of 0.5-2 mg/kg/day until week 2, and then tapered to 5 mg/day at week 24 of treatment. In some embodiments, the method further comprises administering methylprednisolone to the subject by intravenous (IV) infusion at weeks 0, 2, 24, 26, and 52 of treatment, e.g. , prior to administering the type II anti-CD20 antibody. In some embodiments, if the subject weighs greater than or equal to 45 kg, 80 mg of methylprednisolone is administered to the subject. In some embodiments, if the subject weighs less than 45 kg, 1.5 mg/kg of methylprednisolone is administered to the subject.

In certain aspects, provided herein is a kit for treating childhood-onset INS in an individual or reducing the risk and/or frequency of recurrence thereof, comprising: a container comprising a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and the light chain comprising the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6; and a package insert having instructions for use of the type II anti-CD20 in any of the methods described above and herein.

In certain aspects, provided herein is a type II anti-CD20 antibody ( eg , obinutuzumab) for use in any of the methods described above and herein.

It should be understood that one, some or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following embodiments.

Cross-references to related applications

This application claims the benefit of priority to U.S. Provisional Application Serial No. 63/423,767 filed on November 8, 2022, the contents of which are incorporated herein by reference in their entirety. Citation of Electronic Sequence Listing

The contents of the electronic sequence listing (146392064240seqlist.xml; size: 41,773 bytes; and creation date: November 2, 2023) are hereby incorporated by reference in its entirety.

Childhood-onset idiopathic nephrotic syndrome (INS) is also called primary nephrotic syndrome (excluding secondary causes) and encompasses both minimal change disease (MCD) and focal and segmental glomerulosclerosis (FSGS). The disease is usually first diagnosed between the ages of 2 and 5 years (in 70% of patients with MCD), typically affects boys more than girls (2:1), and is defined by the presence of nephrotic-range proteinuria, edema, hyperlipidemia, and hypoalbuminemia (Noone et al. (2018) Lancet 392:61-74). Patients with childhood-onset INS are initially treated with systemic oral corticosteroids. However, childhood-onset INS recurs in more than 75% of patients, and almost 50% of patients demonstrate frequent relapses or steroid dependence (Abdel-Hafez et al. (2017) J. Nephropathol 6:180-186). Despite current treatments, patients remain at significant risk for growth impairment and other significant side effects of steroid-related toxicity. Therefore, there is a continuing need for safer and more effective treatments for childhood-onset INS ( e.g. , FRNS and/or SDNS).

In one aspect, provided herein is a method for treating childhood-onset INS in an individual, comprising administering to the individual at least a first antibody exposure to a Type II anti-CD20 antibody and a second antibody exposure to the Type II anti-CD20 antibody; wherein the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the first antibody exposure comprising: (a) a total exposure of between about 1800 mg and about 2200 mg of the Type II anti-CD20 antibody, or (b) a total exposure of between about 36 mg/kg and about 44 mg/kg of the Type II anti-CD20 antibody, provided that the individual weighs less than 45 kg; wherein the second antibody exposure comprises the Type II anti-CD20 antibody One or two doses of an antibody, the second antibody exposure comprises: (c) a total exposure of between about 1800 mg and about 2200 mg of a Type II anti-CD20 antibody, or (d) a total exposure of between about 36 mg/kg and about 44 mg/kg of a Type II anti-CD20 antibody, provided that the individual weighs less than 45 kg; wherein the Type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprising the HVR-H1 sequence of SEQ ID NO:1, the HVR-H2 sequence of SEQ ID NO:2, and the HVR-H3 sequence of SEQ ID NO:3, and the light chain comprising the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of SEQ ID NO:5, and the HVR-L3 sequence of SEQ ID NO:6 sequence; and wherein the individual is a human who is greater than or equal to 2 years old and less than or equal to 25 years old. In another aspect, provided herein is a method of reducing the risk and/or frequency of relapse in an individual with childhood-onset idiopathic nephrotic syndrome (INS), comprising administering to the individual at least a first antibody exposure to a Type II anti-CD20 antibody and a second antibody exposure to the Type II anti-CD20 antibody; wherein the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the first antibody exposure comprising: (a) a total exposure of between about 1800 mg and about 2200 mg of the Type II anti-CD20 antibody, or (b) between about 36 mg/kg and about 44 mg/kg of the Type II anti-CD20 antibody. wherein the second antibody exposure comprises one or two doses of a Type II anti-CD20 antibody, the second antibody exposure comprising: (c) a total exposure of between about 1800 mg and about 2200 mg of the Type II anti-CD20 antibody, or (d) a total exposure of between about 36 mg/kg and about 44 mg/kg of the Type II anti-CD20 antibody, provided that the individual weighs less than 45 kg; wherein the Type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprising the HVR-H1 sequence of SEQ ID NO:1, the HVR-H2 sequence of SEQ ID NO:2, and the HVR-H3 sequence of SEQ ID NO:3, the light chain comprising the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of SEQ ID NO:5 and the HVR-L2 sequence of SEQ ID NO: 6; and wherein the subject is a human being greater than or equal to 2 years old and less than or equal to 25 years old. I. General Technology

The techniques and procedures described or referenced herein are generally known to those skilled in the art and are commonly performed using conventional methods, for example, the widely used methods described in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel et al., ed. (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual , and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed. (1994)); ed., 1984); Methods in Molecular Biology ，Humana Press； Cell Biology: A Laboratory Notebook (JE Cellis ed., 1998) Academic Press； Animal Cell Culture (RI Freshney ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press； Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell ed., 1993-8) J. Wiley and Sons； Handbook of Experimental Immunology (DM Weir and CC Blackwell ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos ed., 1987); PCR: The Polymerase Chain Reaction (Mullis et al. ed., 1994); Current Protocols in Immunology (JE Coligan et al. ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CAJaneway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, Oxford University Press, 20 00); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by VTDeVita et al., JBLippincott Company, 1993). II. Definition

The term "childhood-onset idiopathic renal syndrome (INS)" refers to idiopathic renal syndromes that are usually first diagnosed between the ages of 2 and 5 years and include minimal change disease (MCD) and focal and segmental glomerulosclerosis (FSGS), also known as primary renal syndromes (when secondary causes are excluded).

The term "antibody" includes monoclonal antibodies (including full-length antibodies with an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies ( such as bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments ( such as Fab, F(ab') ₂ , and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units plus an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2 to 5 basic 4-chain units that can combine with the J chain to form a multivalent combination. For IgG, the 4-chain unit is usually about 150,000 daltons. Each L chain is linked to the H chain by a covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain ( _VH ) at the N-terminus, followed by three constant domains ( _CH ) for each of the α and γ chains, and four _CH domains for the µ and ε isotypes. Each L chain has a variable domain ( _VL ) at the N-terminus, followed by a constant domain at its other end. _{The VL} pairs with the _VH , and the _CL pairs with the first constant domain ( _CH1 ) of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The paired _VH and _VL together form a single antigen-binding site. For the structure and properties of the different classes of antibodies, see , e.g., Basic and Clinical Immunology , 8th ed., Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Based on the amino acid sequence of their constant domain, the L chain from any vertebrate can be classified into one of two clearly distinct types, called kappa (κ) and lambda (λ). Based on the amino acid sequence of their heavy chain constant domain (CH), immunoglobulins can be classified into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with their heavy chains designated α, δ, ε, γ, and µ, respectively. The gamma and alpha classes are further divided into subclasses based on relatively small differences in CH sequence and function, for example , humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains are referred to as " VH " and " VL ", respectively. These domains are usually the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

The term "variable" refers to the fact that certain segments of the variable domain differ greatly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the entire range of the variable domain. Instead, it is concentrated in three segments called highly variable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called the framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, primarily adopting a β-sheet configuration, connected by three HVRs, which form loops connecting the β-sheet structure and in some cases form part of the β-sheet structure. The HVRs in each chain are tightly bound together by the FR regions and together with the HVRs of the other chain form the antigen binding site of the antibody (see Kabat et al., Sequences of Immunological Interest , 5th ed., National Institute of Health, Bethesda, MD (1991)). The homeodomain is not directly involved in the binding of antibodies to antigens, but rather exhibits a variety of effector functions, such as antibody involvement in antibody-dependent cellular cytotoxicity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e. , comprising the same individual antibodies except for possible naturally occurring mutations and/or post-translational modifications ( e.g. , isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, the advantage of monoclonal antibodies is that they are synthesized by fusion tumor cultures and are not contaminated by other immunoglobulins. Therefore, the modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be produced by a variety of techniques, including, for example, the hypodoma method ( e.g. , Kohler and Milstein . , Nature , 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995); Harlow et al. , Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technology (see, e.g., Clackson et al. , Nature , 352: 624-628 (1991); Marks et al. , J. Mol. Biol. 222: 581-597 (1992); Sidhu et al. , J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al ., J. Immunol. Methods 284(1-2): 119-132 (2004); and techniques for producing human or human-like antibodies in animals having partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (see , e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al. , Nature 362: 255-258 (1993); Bruggemann et al. , Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016; Marks et al. , Bio/Technology 10: 779-783 (1992); Lonberg et al. , Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The term "naked antibody" refers to an antibody that is not conjugated with a cytotoxic moiety or a radiolabel.

The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably and refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, a whole antibody includes an antibody having a heavy chain and a light chain including an Fc region. The constant domain can be a native sequence constant domain ( e.g. , a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, a complete antibody can have one or more effector functions.

"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding and/or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; bifunctional antibodies; linear antibodies (see U.S. Pat. No. 5,641,870, Ex. 2; Zapata et al., Protein Eng. 8(10) : 1057-1062 [1995]); single-chain antibody molecules formed from antibody fragments; and multispecific antibodies. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name reflecting the ability to crystallize readily. The Fab fragment consists of the variable region domains ( _VH ) of the entire L chain and H chain and the first constant domain ( _CH1 ) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site. Treatment of the antibody with the enzyme pepsin produces a single large F(ab') ₂ fragment that roughly corresponds to two disulfide-linked Fab fragments that have different antigen binding activities and are still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having additional residues at the carboxyl terminus of the _CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residues of the homeodomains bear a free thiol group. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment contains the carboxyl termini of two H chains linked together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by Fc receptors (FcR) present on certain types of cells.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. The fragment consists of a dimer of one heavy chain and one light chain variable region in tight, non-covalent association. Six highly variable loops (3 loops each from the H and L chains) result from the folding of these two domains, which form the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

"Single-chain Fv", also referred to as "sFv" or "scFv", is an antibody fragment comprising _VH and _VL antibody domains linked in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that enables the sFv to form the desired antigen-binding structure. For a general description of sFv, see Pluckthun's article in The Pharmacology of Monoclonal Antibodies, Vol. 113 (Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315, 1994).

The "functional fragment" of the antibody of the present invention comprises a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The term "diabody" refers to a small antibody fragment prepared by constructing an sFv fragment (see previous paragraph) with a short linker (about 5-10 residues) between the _VH and _VL domains, so that interchain pairing of the fragments of the V domain is achieved, thereby generating a bivalent fragment, i.e. , a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the _VH and _VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail, for example: EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90 : 6444-6448 (1993).

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA , 81 : 6851-6855 (1984)). Chimeric antibodies of interest herein include ^PRIMATIZED® antibodies, in which the antigen binding region of the antibody is derived from an antibody produced, for example , by immunizing a macaque with the target antigen. As used herein, "humanized antibodies" are a subset of "chimeric antibodies."

"Humanized" forms of non-human ( e.g., murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In one embodiment, a humanized antibody is a human immunoglobulin (acceptor antibody) in which HVR (as defined below) residues of the acceptor are replaced by HVR residues of a non-human species (donor antibody), such as mouse, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, the framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may comprise residues that are not present in the acceptor antibody or the donor antibody. These modifications may be performed to further improve antibody properties, such as binding affinity. Typically, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the highly variable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of a portion of the FR region is that of a human immunoglobulin sequence, although the FR region may include one or more single FR residue substitutions that improve antibody performance (such as binding affinity, isomerization, immunogenicity, etc.). The number of these amino acid substitutions in the FR is usually no more than 6 in the H chain and no more than 3 in the L chain. A humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), which is usually a constant region of a human immunoglobulin. For more details, see , e.g. , Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol . 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech . 5: 428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol ., 227: 381 (1991); Marks et al., J. Mol. Biol ., 222: 581 (1991). Methods for preparing human monoclonal antibodies are also described in: Cole et al., Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol. , 147(1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. , 5 : 368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals that have been engineered to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immune transgenic mice (see , e.g. , U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE ^™ technology). For human antibodies produced by human B cell fusion tumor technology, see, for example, Li et al., Proc. Natl. Acad. Sci. USA , 103 : 3557-3562 (2006).

As used herein, the term "hypervariable region", "HVR" or "HV" refers to the region of the variable domain of an antibody whose sequence is highly variable and/or forms a structurally defined loop. In general, an antibody comprises six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the most diversity among the six HVRs, and H3 in particular is believed to play a unique role in conferring superior specificity to antibodies. See, e.g., Xu et al., Immunity 13 : 37-45 (2000); Johnson and Wu, In: Methods in Molecular Biology 248 : 1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, in the absence of light chains, natural camel antibodies consisting only of heavy chains are functional and stable. See, for example: Hamers-Casterman et al., Nature 363 : 446-448 (1993); Sheriff et al., Nature Struct. Biol. 3 : 733-736 (1996).

Many descriptions of HVRs are in use and are covered herein. The Kabat complementarity determining regions (CDRs) are based on sequence variation and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). In contrast, Chothia refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "Contact" HVRs are based on analysis of available complex crystal structures. The residues for each of these HVRs are shown below. Ring Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L 50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat number) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia number) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise an "extended HVR" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. For these definitions, variable domain residues are numbered according to Kabat et al., as described above.

The expression "variable domain residue numbering as described in Kabat" or "amino acid position numbering as described in Kabat" and variants thereof refers to the numbering system for heavy chain variable domains or light chain variable domains used for antibody compilation as described above by Kabat et al . Using this numbering system, the actual linear amino acid sequence may include fewer or additional amino acids corresponding to a shortening or insertion of the FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat numbering) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat numbering). The Kabat numbers of residues in a given antibody can be determined by aligning regions of sequence homology with a "standard" Kabat numbering sequence.

"Backbone" or "FR" residues are those variable domain residues other than the HVR residues as defined herein.

A "human common framework" or "acceptor human framework" is a framework that represents the most common amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences comes from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as described in Kabat et al., Sequences of Proteins of Immunological Interest , 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) . Examples for VL include subgroups κ I, κ II, κ III, or κ IV as described in Kabat et al., supra. In addition, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III, as described in Kabat et al . , supra. Alternatively, a human common framework may be derived from the above, wherein specific residues, for example when a human framework sequence is selected based on its homology to a donor framework by aligning a series of various human framework sequences with a donor framework sequence. An acceptor human framework "derived from" a human immunoglobulin framework or a human common framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence variations. In some embodiments, the number of pre-existing amino acid variations is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

The "VH subgroup III consensus framework" comprises a consensus sequence obtained from the amino acid sequences in the variant subgroup III of Kabat et al. supra . In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (HC-FR1) (SEQ ID NO:35), WVRQAPGKGLEWV (HC-FR2) (SEQ ID NO:36), RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (HC-FR3, SEQ ID NO:37), WGQGTLVTVSA (HC-FR4) (SEQ ID NO:38).

The "VLκI consensus framework" comprises a consensus sequence obtained from the amino acid sequences in the variant light κ subgroup I of Kabat et al. supra . In one embodiment, the VH subgroup I consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: DIQMTQSPSSLSASVGDRVTITC (LC-FR1) (SEQ ID NO:39), WYQQKPGKAPKLLIY (LC-FR2) (SEQ ID NO:40), GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (LC-FR3) (SEQ ID NO:41), FGQGTKVEIKR (LC-FR4) (SEQ ID NO:42).

An "amino acid modification" at a specific position, such as the Fc region, refers to a substitution or deletion of a specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent" to a specified residue refers to an insertion within one to two residues thereof. The insertion may be at the N-terminus or C-terminus of the specified residue. A preferred amino acid modification herein is a substitution.

An "affinity matured" antibody is one that has one or more alterations in one or more of its HVRs that result in an improvement in the affinity of the antibody for the antigen, compared to a parent antibody that does not have these alterations. In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by methods known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describe affinity maturation by shuffling of VH and VL domains. Randomly induced mutagenesis of HVRs and/or backbone residues is described in: Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

As used herein, the term "specifically binds" or "specific for" refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an antigenic determinant) is one that binds to that target with greater affinity, binding, ease, and/or duration than it binds to other targets with greater affinity, binding, ease, and/or duration. In one embodiment, the extent to which the antibody binds to an unrelated target is less than about 10% of the extent to which the antibody binds to the antigen, such as by radioimmunoassay (RIA). In some embodiments, the dissociation constant (Kd) of an antibody that specifically binds to a target is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. In some embodiments, the antibody specifically binds to an antigenic determinant on a protein that is conserved among proteins of different species. In another embodiment, specific binding may include but does not require exclusive binding.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. For example, the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, the composition of the complete antibody may include an antibody population with all K447 residues removed, an antibody population with the K447 residue not removed, and an antibody population having a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc regions for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferably, the FcR is a native sequence human FcR. In addition, preferred FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and splice forms of these receptors, FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. See M. Daëron, Annu. Rev. Immunol. 15 : 203-234 (1997). FcRs are reviewed in: Ravetch and Kinet, Annu. Rev. Immunol. 9 : 457-92 (1991); Capel et al., Immunomethods 4 : 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126 : 330-41 (1995). The term "FcR" herein encompasses other FcRs, including those identified in the future.

The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn which is responsible for the transfer of maternal IgG to the fetus. Guyer et al., J. Immunol. 117 : 587 (1976) and Kim et al., J. Immunol. 24 : 249 (1994). Methods for measuring binding to FcRn are known (see , e.g., Ghetie and Ward, Immunol. Today 18 : (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem . 279 (8): 6213-6 (2004); WO 2004/92219 (Hinton et al.). In vivo FcRn binding and the serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc regions. WO 2004/42072 (Presta) Antibody variants with improved or reduced binding to FcRs are described. See also , e.g., Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001).

As used herein, the phrase "substantially reduced" or "substantially different" refers to a sufficiently high degree of difference between two numerical values (usually one associated with a molecule and the other associated with a reference/comparator molecule) that one skilled in the art would consider the difference between the two values to be statistically significant in the context of the biological characteristic measured by the values ( e.g. , Kd values). For example, the difference between the two values is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50%, based on the value of the reference/comparator molecule.

As used herein, the term "substantially similar" or "substantially identical" means a sufficiently high degree of similarity between two numerical values (e.g., one associated with an antibody of the invention and the other associated with a reference/comparator antibody) such that one skilled in the art would consider the difference between the two values to be small or not biologically and/or statistically significant in the context of the biological property measured by the values ( e.g. , Kd values). For example, the difference between the two values is less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% based on the value of the reference/comparator molecule.

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers are typically pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

“Package insert” means instructions customarily included in commercial drug packages that contain information regarding the indications, usage, dosage, administration, contraindications, other drugs to be used in conjunction with the packaged product, and/or warnings concerning the use of such drugs .

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the treated individual or cell during the course of a clinical pathology. Desired effects of treatment include, but are not limited to, reducing the rate of disease progression, improving or relieving the disease state, reducing or improving prognosis, and delaying disease progression. Delaying the progression of a disease ( e.g. , INS) means delaying, impeding, slowing, retarding, stabilizing, and/or postponing the development of the disease. This delay can be of varying lengths of time, depending on the disease being treated and/or the individual's medical history. As will be apparent to one skilled in the art, a sufficient or significant delay may actually cover prevention because the patient, for example an individual at risk of developing the disease, does not develop the disease.

As used herein, "sustained complete remission" means a response to treatment that includes a first morning fasting UPCR ≤ 0.2 g/g without relapse or any of certain concurrent events ( e.g. , occurring after Week 8), such as (1) relapse as defined by any of the following events requiring systemic corticosteroids or other immunosuppressive therapy: (a) first morning fasting UPCR ≥ 2 g/g or (b) dipstick UA ≥ 3+ for 3 consecutive days with the most recent urine sample during that 3-day period determined to have a UPCR > 0.2 g/g or (c) dipstick UA protein ≥ 3+ with edema on any day with a urine sample determined to have a UPCR > 0.2 g/g; (2) dipstick UA protein ≥ 3+ with edema on any day with a urine sample determined to have a UPCR > 0.2 g/g; (a) use of any systemic corticosteroid for more than 14 days within a 1-day period; (b) initiation of any rescue therapy for INS other than systemic corticosteroids; (c) discontinuation of treatment due to lack of efficacy; or (d) death.

As used herein, "CD20" refers to human B lymphocyte antigen CD20 (also known as CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; the sequence is represented by SwissProt database entry P11836) which is a hydrophobic transmembrane protein with a molecular weight of about 35 kD located on pre-B and mature B lymphocytes. (Valentine, MA et al., J. Biol. Chem. 264(19) (1989 11282-11287; Tedder, TF, et al., Proc. Natl. Acad. Sci. USA 85 (1988) 208-12; Stamenkovic, I., et al., J. Exp. Med. 167 (1988) 1975-80; Einfeld, DA, et al., EMBO J. 7 (1988) 711-7; Tedder, TF, et al., J. Immunol. 142 (1989) 2560-8). The corresponding human gene is transmembrane domain 4, subfamily A, member 1, also known as MS4A1. This gene encodes transmembrane 4A Members of a gene family. Members of this family of nascent proteins are characterized by common structural features and similar intron/exon splice boundaries and display distinct expression patterns between hematopoietic and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in B cell development and differentiation into plasma cells. This family member is located at 11q12, in a cluster of family members. Alternative splicing of this gene results in two transcript variants encoding the same protein.

The terms "CD20" and "CD20 antigen" are used interchangeably herein and include any variants, isoforms, and species homologs of human CD20 that are naturally expressed by cells or expressed on cells transfected with the CD20 gene. Binding of the antibodies of the present invention to the CD20 antigen mediates the killing of cells expressing CD20 (e.g., tumor cells) by inactivating CD20. Toxicity of cells expressing CD20 may occur by one or more of the following mechanisms: cell death/apoptosis induction, ADCC, and CDC.

As recognized in the art, synonyms for CD20 include B lymphocyte antigen CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5.

According to the present invention, the term "anti-CD20 antibody" refers to an antibody that specifically binds to the CD20 antigen. Depending on the binding properties of the anti-CD20 antibody to the CD20 antigen and the biological activity, two types of anti-CD20 antibodies can be distinguished (type I and type II anti-CD20 antibodies), see Cragg, MS, et al., Blood 103 (2004) 2738-2743; and Cragg, MS, et al., Blood 101 (2003) 1045-1052, see Table 1 below. Table 1. Properties of type I and type II anti-CD20 antibodies Type I anti-CD20 antibody Type II anti-CD20 antibody Type I CD20 epitope Type II CD20 epitope Localize CD20 to lipid membrane rafts CD20 is not localized in lipid rafts Increased CDC (if IgG1 isotype) CDC decreased (if IgG1 isotype) ADCC activity (if IgG1 isotype) ADCC activity (if IgG1 isotype) Fully integrated capabilities Reduced binding ability Homotypic aggregation Stronger homotypic aggregation Induction of cell apoptosis during cross-linking Strong cell death induction without cross-linking

Examples of type II anti-CD20 antibodies include, for example, humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody as described in WO 2005/044859), 11B8 IgG1 (as disclosed in WO 2004/035607), and AT80 IgG1. Typically, type II anti-CD20 antibodies of the IgG1 isotype exhibit CDC properties. Compared to type I antibodies of the IgG1 isotype, type II anti-CD20 antibodies have reduced CDC (if IgG1 isotype).

Examples of type I anti-CD20 antibodies include, e.g., rituximab, HI47 IgG3 (ECACC, fusion tumor), 2C6 IgG1 (as disclosed in WO 2005/103081), 2F2 IgG1 (as disclosed in WO 2004/035607 and WO 2005/103081), and 2H7 IgG1 (as disclosed in WO 2004/056312).

The afucosylated anti-CD20 antibody according to the present invention is preferably a type II anti-CD20 antibody, and more preferably an afucosylated humanized B-Ly1 antibody (as described in WO 2005/044859 and WO 2007/031875).

The "rituximab" antibody (reference antibody; example of a type I anti-CD20 antibody) is a genetically engineered chimeric human gamma 1 mouse constant domain containing a monoclonal antibody directed against the human CD20 antigen. However, the antibody is not glycosylated, nor afucosylated, and thus the fucose content is at least 85%. The chimeric antibody contains a human gamma 1 constant domain and is identified by the name "C2B8" in U.S. Patent No. 5,736,137 (Andersen et al.) issued on April 17, 1998 (assigned to IDEC Pharmaceuticals Corporation). Rituximab is approved for the treatment of patients with relapsed or refractory low-grade or follicular CD20-positive B-cell non-Hodgkin's lymphoma. In vitro mechanism of action studies have shown that rituximab exhibits human complement-dependent cytotoxicity (CDC) (Reff, ME et al., Blood 83(2) (1994) 435-445). In addition, it exhibits activity in an assay measuring antibody-dependent cytotoxicity (ADCC).

As used herein, the term "GA101 antibody" refers to any of the following antibodies that bind to human CD20: (1) an antibody comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID No: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (2) an antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7, and a VL domain comprising the amino acid sequence of SEQ ID NO: 8, (3) an antibody comprising the amino acid sequence of SEQ ID NO: 9 and (4) an antibody called obinutuzumab, or (5) an antibody comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 and an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. In one embodiment, the GA101 antibody is an IgG1 isotype antibody. In some embodiments, the anti-CD20 antibody is a humanized B-Ly1 antibody.

The term "humanized B-Ly1 antibody" refers to the humanized B-Ly1 antibody as disclosed in WO 2005/044859 and WO 2007/031875, which is obtained from the murine monoclonal anti-CD20 antibody B-Ly1 (mouse heavy chain variable region (VH): SEQ ID NO: 11; murine light chain variable region (VL): SEQ ID NO: 12 - see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139), which is obtained by chimerization with a human constant domain from IgG1 and subsequent humanization (see WO 2005/044859 and WO 2007/031875). These "humanized B-Ly1 antibodies" are disclosed in detail in WO 2005/044859 and WO 2007/031875. 50 60 Ala Asp Lys Ser Ser Asn Thr Ala Tyr Met Gln Leu Thr Ser Leu Thr 65 70 75 80 Ser Val Asp Ser Ala Val Tyr Leu Cys 75 80 90 100 150 150 200 250 300 300 400 500 450 500 600 700 100 150 200 250 300 400 500 600 700 100 150 200 250 300 400 500 600 700 100 150 200 250 300 400 500 40 45 Leu Val Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr 50 55 60 60 70 80 90 100 110 Variable region of light chain (VL) of murine monoclonal anti-CD20 antibody B-Ly1 (SEQ ID NO: 12) Asn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser 1 5 10 15 Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu 20 25 30 Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn 35 40 45 Leu Val Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr 50 55 60 Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 65 70 75 80 Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly 85 90 95 Thr Lys Leu Glu Ile Lys Arg 100

In one embodiment, the "humanized B-Ly1 antibody" has a variable region of the heavy chain (VH) selected from the group of SEQ ID NOs: 7, 8 and 13 to 33 (in particular corresponding to B-HH2 to B-HH9 and B-HL8 to B-HL17 of WO 2005/044859 and WO 2007/031875). In a specific embodiment, this variable domain is selected from SEQ ID NOs: 14, 15, 7, 19, 25, 27 and 29 (corresponding to B-HH2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11 and B-HL13 of WO 2005/044859 and WO 2007/031875). In a specific embodiment, the "humanized B-Ly1 antibody" has a light chain (VL) variable region of SEQ ID NO: 8 (corresponding to B-KV1 of WO 2005/044859 and WO 2007/031875). In a specific embodiment, the "humanized B-Ly1 antibody" has a heavy chain (VH) variable region of SEQ ID NO: 7 (corresponding to B-HH6 of WO 2005/044859 and WO 2007/031875) and a light chain (VL) variable region of SEQ ID NO: 8 (corresponding to B-KV1 of WO 2005/044859 and WO 2007/031875). Furthermore, in one embodiment, the humanized B-Ly1 antibody is an IgG1 antibody. According to the present invention, these afucosylated humanized B-Ly1 antibodies are glycoengineered (GE) in the Fc region according to the procedures described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180 and WO 99/154342. In one embodiment, the afucosylated glycoengineered humanized B-Ly1 is B-HH6-B-KV1 GE. In one embodiment, the anti-CD20 antibody is obinutuzumab (Recommended INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, obinutuzumab is a synonym for GA101 or RO5072759. This version replaces all previous versions (e.g., Vol. 25, No. 1, 2011, p. 75-76) and was previously known as afutuzumab (Recommended INN, WHO Drug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2, 2008, p. 124). As used herein, reference to obinutuzumab refers to GAZYVA® and its biosimilar antibodies. In some embodiments, the humanized B-Ly1 antibody is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 9, and the light chain comprises the amino acid sequence of SEQ ID NO: 10, or an antigen-binding fragment thereof. In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain variable region comprising three heavy chain CDRs of SEQ ID NO: 9, and a light chain variable region comprising three light chain CDRs of SEQ ID NO: 10. Heavy chain (SEQ ID NO:9) QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA PGQGLEWMGR 50 IFPGDGDTDY NGKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARNV 100 FDGYWLVYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD 150 YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY 200 ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK 250 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV 350 YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 400 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG 449 light chain (SEQ ID NO: 10) DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ 50 LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQNLELP 100 YTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK 150 VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE 200 VTHQGLSSPV TKSFNRGEC                           219

In some embodiments, the humanized B-Ly1 antibody is a glycoengineered humanized B-Ly1 without fucosylation. Such glycoengineered humanized B-Ly1 antibodies have a changed glycosylation pattern in the Fc region, preferably reducing the level of fucosyl residues. Preferably, the amount of fucose is 60% or less of the total amount of oligosaccharides at Asn297 (in one embodiment, the amount of fucose is between 40% and 60%, in another embodiment, the amount of fucose is 50% or less, and in still another embodiment, the amount of fucose is 30% or less). In addition, the oligosaccharides in the Fc region are preferably bisected. These glycoengineered humanized B-Ly1 antibodies have enhanced ADCC.

The ratio of the binding capacity of anti-CD20 antibody to rituximab to CD20 on Raji cells (ATCC-No. CCL-86) was determined by direct immunofluorescence measurement (measurement of mean fluorescence intensity (MFI)) using anti-CD20 antibody conjugated to Cy5 and rituximab conjugated to Cy5, measured with Raji cells (ATCC-No. CCL-86) in FACSArray (Becton Dickinson), as described in Example 2, and was calculated as follows: The ratio of the binding capacity to CD20 on Raji cells (ATCC-No. CCL-86) =

MFI is mean fluorescence intensity. As used herein, "Cy5 labeling rate" refers to the number of Cy5-labeled molecules per antibody molecule.

Typically, the type II anti-CD20 antibody has a ratio of the binding ability of the type II anti-CD20 antibody to rituximab to CD20 on Raji cells (ATCC-No. CCL-86) of 0.3 to 0.6, and in one embodiment 0.35 to 0.55, and in yet another embodiment 0.4 to 0.5.

In one embodiment, the type II anti-CD20 antibody, e.g., GA101 antibody, has increased antibody-dependent cellular cytotoxicity (ADCC).

An "antibody having increased antibody-dependent cellular cytotoxicity (ADCC)" refers to an antibody as defined herein that has increased ADCC as determined by any suitable method known to those of ordinary skill in the art. An acceptable in vitro ADCC assay is as follows: 1) The assay uses target cells known to express the target antigen recognized by the antigen binding region of the antibody; 2) The assay uses human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors as effector cells; 3) The assay is performed according to the following protocol: i) PBMCs are isolated using a standard density centrifugation procedure and suspended in RPMI cell culture medium at a concentration of 5 x ¹⁰⁶ cells/ml; ii) Target cells are grown by standard tissue culture methods, harvested from the exponential growth phase with a viability greater than 90%, washed in RPMI cell culture medium, and washed with 100% PBS. iii) 100 μl of the above final target cell suspension was transferred to each well of a 96-well microtiter plate; iv) the antibody was serially diluted from ⁴⁰⁰⁰ ng/ml to 0.04 ng/ml in cell culture medium and ⁵⁰ μl of the resulting antibody solution was added to the target cells in the 96-well microtiter plate, and various antibody concentrations covering the above concentration range were tested in triplicate; v) for maximum release (MR) controls, an additional 3 μl of the labeled target cells were added to the plate. v) For spontaneous release (SR) controls, 50 μl of RPMI cell culture medium was added to the other 3 wells of the plate containing labeled target cells instead of the antibody solution (point iv above); vii) The 96-well microtiter plate was then centrifuged at 50 × g for 1 min and incubated at 4°C for 1 h; viii) 50 μl of PBMC suspension (point i above) was added to each well to give an effector:target cell ratio of 25:1 and the plate was placed in an incubator with a 5% CO2 atmosphere at 37°C for 4 h; ix) Collect the cell-free supernatant from each well and quantify the experimentally released radioactivity (ER) using a gamma counter; x) Calculate the percentage specific lysis for each antibody concentration according to the formula (ER-MR)/(MR-SR) x 100, where ER is the mean radioactivity quantified for that antibody concentration (see point ix above), MR is the mean radioactivity quantified for the MR control (see point v above), and SR is the mean radioactivity quantified for the SR control (see point vi above); 4) "Increased ADCC" is defined as an increase in the maximum percent specific lysis observed within the antibody concentration range tested above, and/or a decrease in the antibody concentration required to achieve half of the maximum percent specific lysis observed within the antibody concentration range tested above. In one embodiment, the increase in ADCC is relative to ADCC measured by the above assays mediated by the same antibody, produced by the same type of host cells, using the same standard production, purification, formulation, and storage methods known to those skilled in the art, except that the comparator antibody (lacking the increased ADCC) is not produced by host cells engineered to overexpress GnTIII and/or engineered to have reduced expression of a fucosyltransferase 8 (FUT8) gene (e.g., including engineering for a FUT8 knockout).

The "increased ADCC" can be obtained, for example, by mutation and/or glycoengineering of the antibody. In one embodiment, the antibody is glycoengineered to have a bitactinic oligosaccharide attached to the Fc region of the antibody in the GlcNAc bipartite, (e.g., the following: WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.), Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180). In another embodiment, the antibody is glycoengineered to lack fucose on the carbohydrate associated with the Fc region by expressing the antibody in a host cell lacking protein fucosylation (e.g., Lec13 CHO cells or cells in which the α-1,6-fucosyltransferase gene (FUT8) is deleted or the FUT gene expression is knocked down) (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003/085107). In yet another embodiment, the antibody sequence has been engineered in its Fc region to enhance ADCC (e.g., in one embodiment, such an engineered antibody variant comprises an Fc region having one or more amino acid substitutions at positions 298, 333 and/or 334 (EU numbering of residues)).

The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by the antibodies of the present invention in the presence of complement. CDC can be measured by treating a preparation of cells expressing CD20 with an anti-CD20 antibody according to the present invention in the presence of complement. If the antibody induces 20% or more tumor cell lysis (cell death) after 4 hours at a concentration of 100 nM, there is CDC. In one embodiment, the assay is performed with tumor cells labeled with ⁵¹ Cr or Eu and the released ⁵¹ Cr or Eu is measured. Controls include incubating tumor target cells with complement but not with antibody.

The term "expression of CD20" antigen is intended to mean a significant level of expression of the CD20 antigen in a cell, such as a T cell or a B cell. In one embodiment, a patient to be treated according to the method expresses a significant level of CD20 on B cells. CD20 expression on B cells can be determined by standard assays known in the art. For example, expression of the CD20 antigen is detected using an immunohistochemistry (IHC) assay, FACS, or via PCR-based detection of the corresponding mRNA.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a molecule" includes combinations of two or more such molecules and the like.

As used herein, the term "about" refers to the usual error range of each value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself.

It should be understood that the aspects and embodiments of the present invention described herein include "comprising" aspects and embodiments, "consisting of" aspects and embodiments, and "consisting essentially of" aspects and embodiments. III. Methods

In one aspect, provided herein is a method for treating childhood-onset idiopathic nephrotic syndrome (INS) in an individual by administering an effective amount of a type II anti-CD20 antibody; wherein the individual is a human aged 2 years or older and younger than 25 years old. In another aspect, provided herein is a method for reducing the risk and/or frequency of relapse in an individual with childhood-onset idiopathic nephrotic syndrome (INS) by administering an effective amount of a type II anti-CD20 antibody; wherein the individual is a human aged 2 years or older and younger than 25 years old. In one aspect, provided herein is a method for treating childhood-onset INS in an individual or depleting peripheral B cells in an individual with childhood-onset INS by administering an effective amount of a type II anti-CD20 antibody; wherein the individual is a human aged greater than or equal to 2 years and less than or equal to 25 years.

In some embodiments, for example , when the individual weighs greater than or equal to 45 kg, the methods comprise administering to the individual a first antibody exposure to a Type II anti-CD20 antibody and a second antibody exposure to the Type II anti-CD20 antibody, wherein the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the first antibody exposure comprising a total exposure to the Type II anti-CD20 antibody between about 1800 mg and about 2200 mg; and wherein the second antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the second antibody exposure comprising a total exposure to the Type II anti-CD20 antibody between about 1800 mg and about 2200 mg. In some embodiments, e.g. , when the individual weighs less than 45 kg, the methods comprise administering to the individual a first antibody exposure to a Type II anti-CD20 antibody and a second antibody exposure to the Type II anti-CD20 antibody, and providing the second antibody exposure until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the first antibody exposure comprising a total exposure to the Type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg; and wherein the second antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the second antibody exposure comprising a total exposure to the Type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg. As described herein, in some embodiments, the antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3; and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6. In some embodiments, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 7; and a VL domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises an antibody comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, and comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is obinutuzumab. Anti- CD20 Antibodies

Certain aspects of the disclosure relate to anti-CD20 antibodies, for example , for use in the methods described herein, for example, for treating childhood-onset INS ( e.g. , FRNS or SDNS) or reducing the risk and/or frequency of recurrence thereof. In some embodiments, the anti-CD20 antibody is a type II antibody. In some embodiments, the anti-CD20 antibody is human or humanized. In some embodiments, the anti-CD20 antibody is afucosylated. In some embodiments, the anti-CD20 antibody is a GA101 antibody.

Examples of type II anti-CD20 antibodies include, for example, humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody as described in WO 2005/044859), 11B8 IgG1 (as disclosed in WO 2004/035607), and AT80 IgG1. Typically, type II anti-CD20 antibodies of the IgG1 isotype exhibit CDC properties. Compared to type I antibodies of the IgG1 isotype, type II anti-CD20 antibodies have reduced CDC (if IgG1 isotype).

In some embodiments, the anti-CD20 antibody is the GA101 antibody described herein. In some embodiments, the anti-CD20 is any one of the following antibodies that bind to human CD20: (1) an antibody comprising HVR-H1 comprising the amino acid sequence of GYAFSY (SEQ ID NO: 1), HVR-H2 comprising the amino acid sequence of FPGDGDTD (SEQ ID NO: 2), HVR-H3 comprising the amino acid sequence of NVFDGYWLVY (SEQ ID NO: 3), HVR-L1 comprising the amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 4), HVR-L2 comprising the amino acid sequence of QMSNLVS (SEQ ID NO: 5), and HVR-L3 comprising the amino acid sequence of AQNLELPYT (SEQ ID NO: 6); (2) an antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7, and a VL domain comprising the amino acid sequence of SEQ ID NO: 8. domain comprising the amino acid sequence of SEQ ID NO:8, (3) an antibody comprising the amino acid sequence of SEQ ID NO:9 and the amino acid sequence of SEQ ID NO:10; (4) an antibody referred to as obinutuzumab, or (5) an antibody comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:9 and an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:10. In one embodiment, the GA101 antibody is an IgG1 isotype antibody. In some embodiments, the anti-CD20 antibody comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of any antibody described herein, e.g. , 3 HVRs from SEQ ID NO: 7 and 3 HVRs from SEQ ID NO: 8, 3 HVRs from SEQ ID NO: 9 and 3 HVRs from SEQ ID NO: 10, or any HVR of the amino acid sequences provided in Table 2.

In some embodiments, the anti-CD20 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8. QVQLVQSGAEVKKPGSSVKVSCKAS GYAFSY SWINWVRQAPGQGLEWMGRI FPGDGDTD YNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR NVFDGYWLVY WGQGTLVTVSS (SEQ ID NO: 7) DIVMTQTPLSLPVTPGEPASISC RSSKSLLHSNGITYLY WYLQKPGQSPQLLIY QMSNLVS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTV (SEQ ID NO: 8).

In some embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. QVQLVQSGAEVKKPGSSVKVSCKAS GYAFSY SWINWVRQAPGQGLEWMGRI FPGDGDTD YNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR NVFDGYWLVY WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:9) DIVMTQTPLSLPVTPGEPASISC RSSKSLLHSNGITYLY WYLQKPGQSPQLLIY QMSNLVS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC AQNLELPYT FGGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:10)

In some embodiments, the anti-CD20 antibody is a humanized B-Ly1 antibody. In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain variable region comprising three heavy chain CDRs of SEQ ID NO: 9, and a light chain variable region comprising three light chain CDRs of SEQ ID NO: 10. In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 9, and a light chain comprising the sequence of SEQ ID NO: 10.

In some embodiments, the anti-CD20 antibody comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence listed in Table 2 below. Table 2. Polypeptide sequences. CONSTRUCT Peptide sequence SEQ ID NO B-HH1 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSYSWMSWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 13 B-HH2 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWMNWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 14 B-HH3 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWMNWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYLCARNVFDGYWLVYWGQGTLVTVSS 15 B-HH4 QVQLVQSGAEVKKPGASVKVSCKVSGYAFSYSWMNWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 16 B-HH5 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWMSWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 17 B-HH6 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 7 B-HH7 QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWISWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 18 B-HH8 QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWMNWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 19 B-HH9 QVQLVQSGAEVKKPGASVKVSCKASGYTFSYSWMNWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 20 B-HL1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWMHWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS twenty one B-HL2 EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWMHWVQQAPGKGLEWMGRIFPGDGDTDYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATNVFDGYWLVYWGQGTLVTVSS twenty two B-HL3 EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWMNWVQQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCATNVFDGYWLVYWGQGTLVTVSS twenty three B-HL4 QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYSWMSWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS twenty four B-HL8 EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMNWVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 25 B-HL10 EVQLVESGGGLVKPGGSLRLSCAASGFAFSYSWMNWVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 26 B-HL11 QVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMNWVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 27 B-HL12 EVQLVESGAGLVKPGGSLRLSCAASGFTFSYSWMNWVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 28 B-HL13 EVQLVESGGGVVKPGGSLRLSCAASGFTFSYSWMNWVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 29 B-HL14 EVQLVESGGGLKKPGGSLRLSCAASGFTFSYSWMNWVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 30 B-HL15 EVQLVESGGGLVKPGSSLRLSCAASGFTFSYSWMNWVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 31 B-HL16 EVQLVESGGGLVKPGGSLRVSCAASGFTFSYSWMNWVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 32 B-HL17 EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMNWVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS 33 VH signal sequence MDWTWRILFLVAAATGAHS 34 B-KV1 DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTV 8 VL signal sequence MDMRVPAQLLGLLLLWFPGARC 43

In some embodiments, the anti-CD20 antibody ( e.g., type II anti-CD20 antibody) is a glycoengineered antibody without fucosylation. Such glycoengineered antibodies have a change in the glycosylation pattern of the Fc region, preferably reducing the level of fucosyl residues. Preferably, the amount of fucose is 60% or less of the total amount of oligosaccharides at Asn297 (in one embodiment, the amount of fucose is between 40% and 60%, in another embodiment, the amount of fucose is 50% or less, and in still another embodiment, the amount of fucose is 30% or less). In addition, the oligosaccharides in the Fc region are preferably bisected. In some embodiments, the type II anti-CD20 antibody comprises an Fc region comprising a biantennary oligosaccharide bisected by N-acetylglucosamine (GlcNAc). These carbohydrate-engineered humanized anti-CD20 (eg, B-Ly1) antibodies have increased ADCC.

Oligosaccharide content can significantly affect properties relevant to the efficacy of therapeutic glycoproteins, including physical stability, resistance to protease attack, interaction with the immune system, pharmacokinetics, and specific biological activities. Such properties depend not only on the presence or absence of the oligosaccharide, but also on the specific structure of the oligosaccharide. Some generalizations can be made about oligosaccharide structure and glycoprotein function. For example, certain oligosaccharide structures mediate rapid clearance of glycoproteins from the bloodstream by interacting with specific carbohydrate binding proteins, while other oligosaccharide structures may be bound by antibodies and trigger undesirable immune responses. (Jenkins, N., et al., Nature Biotechnol. 14 (1996) 975-81).

Mammalian cells are preferred hosts for the production of therapeutic glycoproteins because of their ability to glycosylate proteins in a form that is most compatible with humans. (Cumming, DA et al., Glycobiology 1 (1991) 115-30; Jenkins, N. et al., Nature Biotechnol. 14 (1996) 975-81). Bacteria rarely glycosylate proteins and, like other types of common hosts (e.g., yeast, filamentous fungi, insects, and plant cells), produce glycosylation patterns that are associated with rapid clearance from the blood, adverse immune interactions, and, in certain circumstances, reduced biological activity. Among mammalian cells, Chinese hamster ovary (CHO) cells have been the most commonly used over the last two decades. In addition to providing the appropriate glycosylation pattern, these cells can consistently generate genetically stable, high-yielding selected cell lines. They can be grown to high densities in simple bioreactors using serum-free media and allow the development of safe and reproducible bioprocesses. Other commonly used animal cells include hamster kidney (BHK) cells, NSO and SP2/0 mouse myeloma cells. Recently, the production of genetically modified animals has also been tested. (Jenkins, N., et al., Nature Biotechnol. 14 (1996) 975-981).

Antibodies may contain carbohydrate structures at conserved positions in the constant region of the heavy chain, with each isotype having a range of different nitrogen-linked carbohydrate structures that may variably affect protein assembly, secretion, or functional activity. (Wright, A., and Morrison, SL, Trends Biotech. 15 (1997) 26-32). The structure of the attached nitrogen-linked carbohydrate varies greatly depending on the degree of processing and can include high mannose, multi-branched, and biantennary complex oligosaccharides. (Wright, A., and Morrison, SL, Trends Biotech. 15 (1997) 26-32). In general, there is heterogeneous processing of the core oligosaccharide structure attached at a specific glycosylation site, so even a single antibody exists in multiple glycoforms. Likewise, it has been shown that major differences in antibody glycosylation occur between cell lines, with even small differences observed for a given cell line grown under different culture conditions (Lifely, MR et al., Glycobiology 5(8) (1995) 813-22).

One approach to achieving greatly improved potency, while maintaining a simple manufacturing process and potentially avoiding significant adverse side effects, is to enhance the natural, cell-mediated effector function of monoclonal antibodies by engineering their oligosaccharide components, as described by Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180 and US 6,602,684. IgG1 type antibodies are the most commonly used antibodies in cancer immunotherapy and are glycoproteins with a conserved N-linked glycosylation site at Asn297 in each CH2 domain. Two complex bitactile oligosaccharides attached to Asn297 are buried between the CH2 domains and form extensive contacts with the polypeptide backbone, and their presence is essential for antibody-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, MR, et al., Glycobiology 5 (1995) 813-822; Jefferis, R., et al., Immunol. Rev. 163 (1998) 59-76; Wright, A., and Morrison, SL, Trends Biotechnol. 15 (1997) 26-32).

It was previously shown that overexpression of β(1,4)-N-acetylglucosamine glycosyltransferase I11 ("GnTII17y"), a glycosyltransferase that catalyzes the formation of bisected oligosaccharides, in Chinese hamster ovary (CHO) cells significantly increased the in vitro ADCC activity of an anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced by engineered CHO cells. (See Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180; and WO 99/154342, the entire contents of which are incorporated herein by reference). Antibody chCE7 GnTIII is a large class of unconjugated monoclonal antibodies that have high tumor affinity and specificity, but when produced in standard industrial cell lines lacking the GnTIII enzyme, the titers are too low to be clinically useful (Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180). This study shows for the first time that ADCC activity can be greatly enhanced by engineering antibody-producing cells to produce GnTIII, which also results in an increase in the proportion of constant region (Fc)-associated bipartite oligosaccharides (including bipartite non-fucosylated oligosaccharides) above the level found in natural antibodies.

In some embodiments, the anti-CD20 antibody ( e.g., type II anti-CD20 antibody) comprises a human Fc region ( e.g., a human IgG1 Fc region). In some embodiments, the Fc region comprises nitrogen-linked oligosaccharides that have been modified. In some embodiments, the nitrogen-linked oligosaccharides of the Fc region have reduced fucosylation residues compared to antibodies having unmodified nitrogen-linked oligosaccharides. In some embodiments, the bisected oligosaccharides are bisected complex oligosaccharides. In some embodiments, the nitrogen-linked oligosaccharides have been modified to have increased bisected, non-fucosylated oligosaccharides. In some embodiments, the bisected, non-fucosylated oligosaccharides are of a mixed type. In some embodiments, the bipartite, non-fucosylated oligosaccharide is of the complex type. For a more detailed description, see, for example , WO 2003/011878 (Jean-Mairet et al. ); U.S. Patent No. 6,602,684 (Umana et al .); US 2005/0123546 (Umana et al. ); and U.S. Patent No. 8,883,980 (Umana et al. ).

In some embodiments , the type II anti-CD20 antibody is obinutuzumab.

The antibodies according to any of the above embodiments ( eg , the type II anti-CD20 antibodies of the present disclosure) may incorporate any of the characteristics described in Sections 1-7 below, either alone or in combination: 1. Antibody affinity

In certain embodiments, the dissociation constant (Kd) of an antibody provided herein is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ , e.g., 10 ⁻⁹ M to 10 ⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed with a Fab form of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of ( ^125I )-labeled antigen in the presence of a serial series of unlabeled antigens and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions for the assay, ^MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate, pH 9.6, and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In nonadsorbent plates (Nunc #269620), 100 pM or 26 pM [ ^125I ]-antigen was mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 described by Presta et al., Cancer Res. 57: 4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation may be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., for 1 hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 ^® ) in PBS. When the plate is dry, scintillator (MICROSCINT-20 ^TM ; Packard) is added at 150 μl/well and counted for ten minutes using a TOPCOUNT ^TM Gamma Counter (Packard). Concentrations of each Fab that provide less than or equal to 20% of the maximum binding concentration are selected for use in competitive binding assays.

According to another embodiment, Kd is measured using a ^BIACORE® surface plasmon resonance assay. For example, the assay is performed using a BIACORE® ^- 2000 or BIACORE® ^- 3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip at about 10 reaction units (RU). In one aspect, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N -ethyl- N' -(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μg/ml (approximately 0.2 μM) in 10 mM sodium acetate (pH 4.8) and injected at a flow rate of 5 μl/min to obtain approximately 10 reaction units (RU) of coupled protein. After injection of antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected in PBS containing 0.05% polysorbate 20 (TWEEN-20 ^TM ) surfactant (PBST) at 25°C at a flow rate of approximately 25 μl/min. The association rate (kon) and dissociation rate (koff) were calculated by simultaneously fitting the binding and dissociation sensor spectra using a simple one-to-one Langmuir binding model ( ^BIACORE® Evaluation Software Version 3.2). The equilibrium dissociation constant (Kd) was calculated from the ratio of koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the binding rate measured by the surface plasmon resonance assay above exceeds 106 M-1 s-1, the binding rate can be determined using the fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25oC, in the presence of increasing concentrations of antigen, as measured in a spectrophotometer such as a stopped flow spectrophotometer (Aviv Instruments) or a stirred cuvette 8000 series SLM-AMINCO ^TM spectrophotometer (ThermoSpectronic). 2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies , Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising salvage receptor binding antigenic determinant residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

Bifunctional antibodies are antibody fragments with two antigen binding sites (which may be bivalent or bispecific). See, e.g., EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

A single domain antibody is an antibody fragment that comprises all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1 ).

Antibody fragments can be produced by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies as described herein and production in recombinant host cells (e.g., E. coli or bacteriophage). 3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al ., Proc. Natl. Acad. Sci. USA , 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed compared to its parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized antibodies to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies comprise one or more variable domains, wherein HVRs such as CDRs (or portions thereof) are derived from non-human antibodies, and FRs (or portions thereof) are derived from human antibody sequences. Humanized antibodies will optionally comprise at least a portion of a human constant region. In certain embodiments, some FR residues in humanized antibodies are substituted with corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are generally described in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, e.g., Riechmann et al. , Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (specifically describing SDR grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling");Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer , 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best match" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of a particular subset of light chain or heavy chain variable regions of human antibodies (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA , 89:4285 (1992); and Presta et al. J. Immunol. , 151:2623 (1993)); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from a screened FR library (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997); and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996). 4. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in: van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001); and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic challenge. These animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or are present extrachromosomally or randomly integrated into the chromosomes of the animal. In these transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example: U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE ^™ technology); U.S. Patent No. 5,770,429 (describing HuMab® technology); U.S. Patent No. 7,041,870 (describing KM MOUSE® technology); and U.S. Patent Application Publication No. US 2007/0061900 (describing VelociMouse® technology). The human variable regions from intact antibodies generated by these animals can be further modified, for example, by combining with different human constant regions.

Human antibodies can also be prepared by fusion tumor-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol. , 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol ., 147: 86 (1991).) Human antibodies produced by human B cell fusion tumor technology are also described in Li et al ., Proc. Natl. Acad. Sci. USA , 103: 3557-3562 (2006). Other methods include those described in, for example, U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies by hybridoma cell lines), and Ni, Xiandai Mianyixue , 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in the following references: Vollmers and Brandlein, Histology and Histopathology , 20(3):927-937 (2005); and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology , 27(3):185-91 (2005).

Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human phage display libraries. These variable domain sequences can then be combined with the desired human constant domains. The following describes techniques for selecting human antibodies from antibody libraries. 5. Antibodies from libraries

Antibodies for use in the present invention can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. For example, various methods known in the art are used to generate phage display libraries and screen such libraries for antibodies having desired binding properties. Such methods are summarized in, e.g., Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described in, e.g., McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al . J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, and then antigen-binding phage can be screened according to the methods described in the following references: Winter et al., Ann. Rev. Immunol. , 12: 433-455 (1994). Phage usually display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources can provide high-affinity antibodies to the immunogen without the need to construct fusion tumors. Alternatively, natural repertoires (e.g., from humans) can be cloned without any immunization to provide a single source of antibodies to a variety of non-self and self antigens, as described by Griffiths et al. in EMBO J, 12: 725-734 (1993). Finally, natural repertoires can be synthesized by selecting unrearranged V gene segments in stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve rearrangement in vitro , as described by Hoogenboom and Winter in J. Mol. Biol. , 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are referred to herein as human antibodies or human antibody fragments .

In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for CD20, while the other specificity is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different antigenic determinants of CD20. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CD20. Bispecific antibodies can be made as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic manipulation to make antibody Fc-heterodimer molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science , 229: 81 (1985)); using leucine zipper to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. , 148(5):1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al. , Proc. Natl. Acad. Sci. USA , 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al. , J. Immunol. , 152:5368 (1994)); and preparing trispecific antibodies as described in Tutt et al., J. Immunol. 147:60 (1991).

Also included herein are engineered antibodies with three or more antigen binding sites, including "Octopus antibodies" (see, e.g., US 2006/0025576A1).

The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs," which contain an antigen binding site that binds to CD20 and another different antigen (see, for example, US 2008/0069820). 7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding characteristics. a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution-induced mutagenesis include HVRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table A. More substantial variations are provided under the heading "Exemplary Substitutions" in Table A and are further described below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into target antibodies and the products screened for desired activity, e.g., retained/improved antigen binding characteristics, reduced immunogenicity, or improved ADCC or CDC. Table A Original Residue Exemplary substitutions Better replacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn；Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; nor-leucine Leu Leu (L) nor-leucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; nor-leucine Leu

Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions entail exchanging a member of one of these classes for a member of another class.

One type of substitutional variant involves replacing one or more highly variable region residues of a parent antibody ( e.g. , a humanized or human antibody). Typically, the resulting variant selected for further study will have modifications (e.g., improvements) and/or substantially retain certain biological properties of the parent antibody (e.g., increased affinity, reduced immunogenicity). Exemplary substitutional variants are affinity-matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display, such as those described herein. In brief, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activities (e.g., binding affinity).

Changes (e.g., substitutions) can be made in HVRs to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that undergo high frequency mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and/or residues that contact the antigen, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing secondary libraries and reselecting therefrom has been described, e.g., in Hoogenboom et al. , Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variant genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A second library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity is the HVR directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues that participate in antigen binding can be specifically identified by, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be implemented in HVRs. For example, such modifications may be outside of antigen contact residues in HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged or contains no more than one, two or three amino acid substitutions.

A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science , 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at the amino acid position, demonstrating good functional sensitivity to the initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antibody and the antigen. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions to the N- or C-terminus of the antibody to enzymes (e.g., for ADEPT) or polypeptides that increase the serum half-life of the antibody. b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibodies are glycosylated. Addition or deletion of glycosylation sites in an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

When the antibody comprises an Fc region, the carbohydrates associated therewith may be altered. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides that are typically attached to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides may include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention may be modified to produce antibody variants having certain improved properties.

In one embodiment, antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The content of fucose relative to the sum of all carbohydrate structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry is determined by calculating the average content of fucose in the Asn297 carbohydrate chain, for example, as described in WO 2008/077546. Asn297 refers to the asparagine residue located near position 297 of the Fc region (Eu numbering of residues in the Fc region); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300 due to minor sequence variations of the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al ., especially in Example 11); and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng , 94(4):680-688 (2006); and WO2003/085107).

Further provided are antibody variants having bisected oligosaccharides, for example, wherein a bi-antenna oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al .). Antibody variants having at least one galactose residue attached to the Fc region on the oligosaccharide are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). c) Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In certain embodiments, the present invention relates to antibody variants that possess some but not all effector functions, making them ideal drug candidates for applications where the in vivo half-life of the antibody is important and certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells that mediate ADCC, NK cells, express only Fc(RIII), whereas monocytes express Fc(RI, Fc(RII, and Fc(RIII). The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of the article by Ravetch and Kinet ( Annu. Rev. Immunol. 9:457-492 (1991)). Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 83 :7059-7063 (1986)). 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox ^96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be measured, for example, in an animal model (e.g., Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998)). A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3c binding ELISAs. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al. , J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn Binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those in which one or more of the Fc region residues 238, 265, 269, 270, 297, 327 and 329 are substituted (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

In certain embodiments, the Fc variants described herein further comprise one or more amino acid modifications (e.g., CDC and/or ADCC) for reducing effector function. In exemplary embodiments, the modification that reduces effector function is a modification that does not change the glycosylation pattern of the Fc region. In certain embodiments, the modification that reduces effector function reduces or eliminates binding to human effector cells, binding to one or more Fc receptors, and/or binding to cells expressing Fc receptors. In an exemplary embodiment, the Fc variants described herein comprise the following modifications: L234A, L235A, and P329G in the human IgG1 Fc region, resulting in reduced effector function. Substitutions L234A, L235A, and P329G (the L234A/L235A/P329G triple variant is referred to as LALAPG) have been previously shown to reduce binding to Fc receptors and complements (see, e.g., U.S. Publication No. 2012/0251531).

In various embodiments, an Fc variant with reduced effector function refers to an Fc variant that reduces effector function (e.g., activities such as CDC, ADCC, and/or binding to FcR) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more compared to the effector function achieved by a wild-type Fc region (e.g., an Fc region that does not have a mutation that reduces effector function, although it may have other mutations). In certain embodiments, an Fc variant with reduced effector function refers to an Fc variant that eliminates all detectable effector function compared to a wild-type Fc region. Assays for measuring effector function are known in the art and are described below.

Cytotoxicity assays in vitro and/or in vivo can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) binding capacity. Primary cells that mediate ADCC NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in the following: Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox ^96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally , ADCC activity of the target molecule may be assessed in vivo in an animal model such as that disclosed in Clynes et al ., Proc. Natl Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al. , J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).

Certain antibody variants with improved or reduced binding to FcRs have been described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333 and/or 334 (EU numbering of residues) of the Fc region.

In some embodiments, modifications are made in the Fc region resulting in modified ( ie, improved or reduced) C1q binding and/or complement-dependent cytotoxicity (CDC), such as described in U.S. Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with longer half-lives and improved binding to the neonatal Fc receptor (FcRn) (which is responsible for the transfer of maternal IgG to the fetus, see Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 for other examples of Fc region variants. d) Cysteine-engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties (e.g., drug moieties or linker-drug moieties) to form immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the Fc region of the heavy chain. Cysteine engineered antibodies can be produced, for example, according to the method described in U.S. Patent No. 7,521,541. e) Antibody Derivatives

In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known in the art and readily available. Suitable derivatized moieties for antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-triazine, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymer used for derivatization may be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under specified conditions, etc.

In another embodiment, a complex of an antibody and a non-protein portion is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein portion is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605, 2005). The radiation can be of any wavelength and includes, but is not limited to, a wavelength that does not damage normal cells but heats the non-protein portion to a temperature close to that at which cells of the antibody-non-protein portion are killed. A. Recombination Methods and Compositions

Antibodies can be made using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, isolated nucleic acids encoding anti-CD20 antibodies as described herein are provided. Such nucleic acids encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and/or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, host cells comprising such nucleic acids are provided. In this embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method for preparing an anti-CD20 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as described above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

When the anti-CD20 antibody is produced recombinantly, nucleic acids encoding the antibody, such as those described above, are isolated and inserted into one or more vectors for further propagation and/or expression in host cells. Such nucleic acids can be easily isolated and sequenced by known methods (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly where glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli .) Following expression, the antibodies can be separated from the soluble portion of the bacterial cell paste and can be further purified .

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable hosts for the cloning or expression of antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of antibodies with partially or fully human glycosylation patterns. See: Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of bacilliform virus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See , e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES ^™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell strains adapted to growth in suspension can be used. Other examples of mammalian host cell lines that can be used include: monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse testicular Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described by Mather et al., Annals NY Acad. Sci . 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2/0. For a review of some mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology , Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). B. Assay

Various assays known in the art can be used to identify, screen or characterize the physical/chemical properties and/or biological activities of the anti-CD20 antibodies provided herein. 1. Binding Assays and Other Assays

In one aspect, the antigen binding activity of the antibodies of the invention is tested, for example, by known methods such as ELISA, Western blot, etc. CD20 binding can be determined using methods known in the art, and exemplary methods are disclosed herein. In one embodiment, binding is measured using a radioimmunoassay. An exemplary radioimmunoassay is provided below. The CD20 antibody is iodinated, and a competitive reaction mixture is prepared, comprising a fixed concentration of the iodinated antibody and a decreasing concentration of a serially diluted unlabeled CD20 antibody. Cells expressing CD20 (e.g., BT474 cells stably transfected with human CD20) are added to the reaction mixture. After incubation, the cells are washed to separate free iodinated CD20 antibodies from CD20 antibodies bound to the cells. The level of bound iodinated CD20 antibodies is determined, for example, by counting the radioactivity associated with the cells, and the binding affinity is determined using standard methods. In another embodiment, the ability of the CD20 antibodies to bind to surface expressed CD20 (e.g., on a subpopulation of B cells) is assessed using flow cytometry. Peripheral leukocytes (e.g., from humans, cynomolgus monkeys, rats, or mice) are obtained and the cells are blocked with serum. Labeled CD20 antibodies are added in serial dilutions, and T cells are also stained to identify T cell subpopulations (using methods known in the art). After incubation and washing of the sample, cells are sorted using flow cytometry and the data analyzed using methods well known in the art. In another embodiment, surface plasmon resonance can be used to analyze CD20 binding. An exemplary surface plasmon resonance method is illustrated in the Examples.

In another aspect, competition assays can be used to identify antibodies that compete with any of the anti-CD20 antibodies disclosed herein for binding to CD20. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as any of the anti-CD20 antibodies disclosed herein. Detailed exemplary methods for mapping antigenic determinants bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized CD20 is incubated in a solution containing a first labeled antibody (e.g., rituximab, GA101 antibody, etc.) that binds to CD20 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to CD20. The second antibody may be present in the fusion tumor supernatant. As a control, immobilized CD20 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to CD20, excess unbound antibody is removed and the amount of label associated with immobilized CD20 is measured. If the amount of label associated with immobilized CD20 in the test sample is significantly reduced relative to the control sample, this indicates that the secondary antibody is competing with the primary antibody for binding to CD20. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 2. Activity Assay

The anti-CD20 antibodies ( eg, type II antibodies) disclosed herein can be identified and/or characterized by one or more activity assays known in the art. For example, complement-dependent cytotoxicity (CDC) and/or antibody-dependent cytotoxicity (ADCC) can be used as described herein.

It will be appreciated that any of the above assays may be performed using the immunoconjugates of the invention in place of or in addition to anti-CD20 antibodies.

It should be understood that any of the above assays can be performed using an anti-CD20 antibody and an additional therapeutic agent. Methods of Administering Type II Anti -CD20 Antibodies

Provided herein are methods of treating childhood-onset idiopathic nephrotic syndrome (INS) in a subject, wherein the method comprises administering to the subject a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody. Also provided herein are methods of depleting circulating peripheral B cells in a subject, wherein the method comprises administering to the subject a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody, and wherein after administration of the type II anti-CD20 antibody, B cells are depleted to a level such that circulating peripheral B cells are present in peripheral blood from the subject at about 5 cells/µL or less. Also provided herein are methods for depleting circulating peripheral B cells in an individual, wherein the method comprises administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody, and wherein, after administration of the type II anti-CD20 antibody, B cell depletion is achieved to a level of circulating peripheral B cells present in the individual's peripheral blood of about 5 cells per μL or less, which persists for at least 52 weeks after the first dose of the first antibody exposure. In some embodiments of the methods herein, the individual or patient is a human being greater than or equal to 2 years old and less than or equal to 25 years old. In some embodiments, the individual or patient is a human who is older than 2 years old and younger than 25 years old. In some embodiments, for example , using weight-based dosing of a type II anti-CD20 antibody, the individual weighs less than 45 kg. In some embodiments, for example , using fixed dosing of a type II anti-CD20 antibody, the individual weighs greater than or equal to 45 kg.

In some embodiments, the individual or patient has been diagnosed with INS ( e.g. , FRNS or SDNS) before the age of 18. Guidelines for the diagnosis of childhood-onset INS ( e.g. , FRNS or SDNS) are known in the art and include, but are not limited to, those described in Kidney Disease: Improving Global Outcomes Glomerular Diseases Work Group. KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases ( Kidney Int. 2021; 100: S1-276).

In some embodiments, the individual or patient has Frequently Relapsing Nephrotic Syndrome (FRNS) of childhood onset. In some embodiments, the individual or patient has ≥2 relapses every 6 months within 6 months of disease onset, or ≥4 relapses every 12 months during any subsequent 12-month period.

In some embodiments, the individual or patient has childhood-onset steroid-dependent nephrotic syndrome (SDNS). In some embodiments, the individual or patient has two consecutive relapses during treatment with prednisone or penicillin (at full dose or during a taper) or within 15 days of cessation of prednisone or penicillin.

In some embodiments, the individual or patient is in complete remission, e.g. , prior to treatment with the methods of the present disclosure. In some embodiments, complete remission is defined as absence of edema, UPCR ≤ 0.2 g/g, and urine dipstick readings of trace or negative for protein for 3 consecutive days.

In some embodiments, the individual or patient has experienced at least 1 relapse within 6 months ( eg , prior to treatment according to the methods of the disclosure).

In some embodiments, the individual or patient has received cyclophosphamide within 6 months ( eg , prior to treatment according to the methods of the present disclosure) and has experienced at least 1 relapse following discontinuation of cyclophosphamide.

In some embodiments, the individual or patient has an estimated glomerular filtration rate (eGFR) within a normal range for their age.

In some embodiments, the methods of the disclosure comprise administering to the individual a first antibody exposure to a type II anti-CD20 antibody of the disclosure and a second antibody exposure to a type II anti-CD20 antibody of the disclosure. In some embodiments, the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not provided until about 18 weeks after the first antibody exposure, about 19 weeks after the first antibody exposure, about 20 weeks after the first antibody exposure, about 21 weeks after the first antibody exposure, about 22 weeks after the first antibody exposure, about 23 weeks after the first antibody exposure, about 24 weeks after the first antibody exposure, about 25 weeks after the first antibody exposure, or about 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not provided until less than about any of the following weeks after the first antibody exposure: 26, 25, 24, 23, 22, 21, 20, or 19. In some embodiments, the second antibody exposure is not provided until greater than about any of the following weeks after the first antibody exposure: 18, 19, 20, 21, 22, 23, 24, or 25. That is, the second antibody exposure is not provided until any one of a range of weeks having an upper limit of 26, 25, 24, 23, 22, 21, 20, or 19 and an independently selected lower limit of 18, 19, 20, 21, 22, 23, 24, or 25, wherein the lower limit is less than the upper limit.

The dosing regimens described herein use a consistent system to track the time between doses, whereby a first dose is administered to a patient on Day 1 or Week 0. As described herein, the antibody exposure of the present disclosure may include one dose or two doses. In the case where the antibody exposure comprises one dose, reference to providing a second antibody exposure after a period of time after the first antibody exposure (as described herein) refers to the amount of time between the dose of the first antibody exposure ( e.g. , Day 1 or Week 0) and the dose of the second antibody exposure. If the first antibody exposure comprises two doses, the first dose of the first antibody exposure is provided on Day 1 or Week 0. In the case where the antibody exposure comprises one dose, reference to providing a second antibody exposure after a period of time after a first antibody exposure (as described herein) refers to the amount of time between the first dose of two doses of the first antibody exposure ( e.g., Day 1 or Week 0) and the first dose of two doses of the second antibody exposure. For example, if the method of the disclosure comprises two doses of a first antibody exposure and two doses of a second antibody exposure, and the second antibody exposure is not provided until about 22 weeks after the first antibody exposure, then the first dose of the first antibody exposure and the first dose of the second antibody exposure are about 22 weeks.

In some embodiments, the first antibody exposure of the present disclosure comprises one or two doses of a Type II anti-CD20 antibody of the present disclosure. In some embodiments, the first antibody exposure comprises a single dose of between about 1800 mg and about 2200 mg of the Type II anti-CD20 antibody. In some embodiments, the first antibody exposure comprises a total exposure of between about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, or about 2200 mg of the Type II anti-CD20 antibody. In some embodiments, the individual weighs greater than or equal to 45 kg.

In some embodiments, the first antibody exposure comprises a total exposure of between about 36 mg/kg and about 44 mg/kg of Type II anti-CD20 antibody. In some embodiments, the first antibody exposure comprises a total exposure of about 36 mg/kg, about 38 mg/kg, about 40 mg/kg, about 42 mg/kg, or about 44 mg/kg of Type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

In some embodiments, the first antibody exposure comprises two doses. In some embodiments, the first antibody exposure comprises a first dose of between about 900 mg and about 1100 mg of the type II anti-CD20 antibody and a second dose of between about 900 mg and about 1100 mg of the type II anti-CD20 antibody. In some embodiments, the first dose of the first antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the individual weighs greater than or equal to 45 kg.

In some embodiments, the first antibody exposure comprises two doses. In some embodiments, the first antibody exposure comprises a first dose of a Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and a second dose of a Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg. In some embodiments, the first dose of the first antibody exposure contains about 20 mg/kg of Type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure contains about 20 mg/kg of Type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

In some embodiments, the second dose of the first antibody exposure is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the first antibody exposure. In some embodiments, the second dose of the first antibody exposure is not provided until about 2 weeks after the first dose of the first antibody exposure.

In some embodiments, the second antibody exposure of the present disclosure comprises one or two doses of a Type II anti-CD20 antibody of the present disclosure. In some embodiments, the second antibody exposure comprises a total exposure of between about 1800 mg and about 2200 mg of a Type II anti-CD20 antibody. In some embodiments, the second antibody exposure comprises a total exposure of between about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, or about 2200 mg of the Type II anti-CD20 antibody. In some embodiments, the individual weighs greater than or equal to 45 kg.

In some embodiments, the second antibody exposure comprises a total exposure of between about 36 mg/kg and about 44 mg/kg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure comprises a total exposure of about 36 mg/kg, about 38 mg/kg, about 40 mg/kg, about 42 mg/kg, or about 44 mg/kg of type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

In some embodiments, the second antibody exposure comprises two doses. In some embodiments, the second antibody exposure comprises a first dose of between about 900 mg and about 1100 mg of the type II anti-CD20 antibody and a second dose of between about 900 mg and about 1100 mg of the type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the individual weighs greater than or equal to 45 kg.

In some embodiments, the second antibody exposure comprises two doses. In some embodiments, the second antibody exposure comprises a first dose of a Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and a second dose of a Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg. In some embodiments, the first dose of the second antibody exposure contains about 20 mg/kg of Type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure contains about 20 mg/kg of Type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

In some embodiments, the second dose of the second antibody exposure is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the second antibody exposure. In some embodiments, the second dose of the second antibody exposure is not provided until about 2 weeks after the first dose of the second antibody exposure.

In some embodiments, the type II anti-CD20 antibodies of the present disclosure are administered intravenously ( eg, by IV infusion).

In some embodiments, the methods of the present disclosure further comprise administering an effective amount of a glucocorticoid or corticosteroid ( e.g. , in combination with a type II anti-CD20 antibody described herein). A variety of naturally occurring and synthetic glucocorticoids/corticosteroids are known in the art, including but not limited to beclometasone, triamcinolone, dexamethasone, betamethasone, prednisone, methylprednisolone, prednisolone, corticosterone, and corticol. In some embodiments, the glucocorticoid or corticosteroid comprises methylprednisolone. In some embodiments, the glucocorticoid or corticosteroid comprises prednisone. The effective amount of the glucocorticoid or corticosteroid disclosed herein is known in the art and can be easily determined by standard assays. For example, methylpernicorticol can be administered once daily by IV in a dose of 750-1000 mg. As another example, prednisone can be administered orally at 0.5 mg/kg and gradually reduced to 7.5 mg per day as appropriate. In some embodiments, methylpernicorticol can be administered before each anti-CD20 antibody infusion. In some embodiments, methylpernicorticol can be administered intravenously at 80 mg ( e.g. , if the individual weighs more than or equal to 45 kg) or 1.5 mg/kg ( e.g. , if the individual weighs less than 45 kg). In some embodiments, oral prednisone or an equivalent may be administered at a dose of 0.5-1 mg/kg/day (maximum 60 mg/day). In some embodiments, oral prednisone or an equivalent may be administered at a dose of 0.5-1 mg/kg/day (maximum 60 mg/day) and gradually reduced to a target of 5 mg/day. In some embodiments, oral prednisone or an equivalent may be administered at a dose of 0.5-2 mg/kg/day (maximum 60 mg/day). In some embodiments, oral prednisone or an equivalent may be administered at a dose of 0.5-2 mg/kg/day (maximum 60 mg/day) and gradually reduced to a target of 5 mg/day.

In some embodiments, a glucocorticoid may be administered before, during, or after administration of a type II anti-CD20 antibody of the present disclosure. In some embodiments, a glucocorticoid may be administered before administration of a type II anti-CD20 antibody of the present disclosure, for example , 30 to 60 minutes before administration of a type II anti-CD20 antibody of the present disclosure. In some embodiments, 80 mg of methylprednisolone may be administered by IV 30 to 60 minutes before administration of a type II anti-CD20 antibody of the present disclosure. In some embodiments, prednisone ( e.g. , administered orally) and/or methylprednisolone ( e.g. , administered IV) may be administered, followed by maintenance therapy ( e.g. , pyroptidine or cyclophosphamide).

In some embodiments, the methods disclosed herein further comprise administering an effective amount of an antihistamine ( e.g. , in combination with a type II anti-CD20 antibody described herein). Antihistamines known in the art and currently used clinically include histamine _H1 receptor and histamine _H2 receptor antagonists or inverse agonists. In some embodiments, the antihistamine comprises diphenhydramine. The effective amount of the antihistamine disclosed herein is known in the art and can be easily determined by standard assays. For example, diphenhydramine can be administered in an oral dose of 0.5-1 mg/kg (rounded to the nearest available pill formulation), to a maximum dose of 50 mg.

In some embodiments, antihistamine can be administered before, during, or after administration of a type II anti-CD20 antibody of the disclosure, for example, as a pre-treatment. In some embodiments, antihistamine can be administered before administration of a type II anti-CD20 antibody of the disclosure, for example , 30 to 60 minutes before administration of a type II anti-CD20 antibody of the disclosure. In some embodiments, 0.5-1 mg/kg or up to 50 mg of diphenhydramine can be administered orally 30 to 60 minutes before administration of a type II anti-CD20 antibody of the disclosure.

In some embodiments, the method of the present disclosure further comprises administering an effective amount of acetaminophen. For example, acetaminophen can be administered at an oral dose of 15 mg/kg, up to a maximum dose of 1000 mg.

In some embodiments, acetaminophen can be administered before, during, or after administration of a type II anti-CD20 antibody of the disclosure, for example as a pre-treatment. In some embodiments, acetaminophen can be administered before administration of a type II anti-CD20 antibody of the disclosure, for example 30 to 60 minutes before administration of a type II anti-CD20 antibody of the disclosure. In some embodiments, 15 mg/kg (rounded to the nearest available pill formulation) or up to 1000 mg of acetaminophen can be administered orally 30 to 60 minutes before administration of a type II anti-CD20 antibody of the disclosure.

In some embodiments, the methods of the present disclosure further comprise administering a standard of care therapy ( e.g. , in combination with a type II anti-CD20 antibody described herein). In some embodiments, a standard of care therapy may be administered before, during, or after administration of a type II anti-CD20 antibody of the present disclosure, e.g. , to treat or prevent one or more symptoms of INS.

In some embodiments, the methods of the disclosure result in complete remission in a subject. In some embodiments, the subject is in complete remission 1 year after initiation of treatment, e.g. , at week 52, as described herein. In some embodiments, complete remission refers to a state of having a first morning fasting UPCR ≤ 0.2 g/g after completion of steroid taper and without complications (occurring after week 8), e.g. , as described herein. In some embodiments, complete remission is sustained, e.g. , 1 year after initiation of treatment, e.g. , at week 52, as described herein. In some embodiments, sustained complete remission includes a first morning fasting UPCR ≤ 0.2 g/g and the absence of any of the following concurrent events: (1) relapse ( e.g. , occurring after Week 8), as defined by any of the following events requiring systemic corticosteroids or other immunosuppressive therapy: (a) first morning fasting UPCR ≥ 2 g/g or (b) 3 consecutive days of dipstick UA ≥ 3+ with a UPCR > 0.2 g/g determined in the most recent urine sample during the 3-day period or (c) dipstick UA protein ≥ 3+ with edema on any day with a UPCR > 0.2 g/g determined in the urine sample; (2) any systemic corticosteroid use for more than 14 days within a 30-day period ( e.g. , on Week 8 weeks later); (3) initiation of any rescue therapy for INS other than systemic corticosteroids ( e.g. , occurring at any time); (4) discontinuation of treatment due to lack of efficacy ( e.g. , occurring at any time); or (5) death ( e.g. , occurring at any time). In some embodiments, the subject is in sustained complete remission from Week 8 to Week 52 of treatment, and the subject has not experienced (1) a relapse, as defined by any of the following events requiring systemic corticosteroids or other immunosuppressive therapy: (a) first morning fasting UPCR ≥ 2 g/g or (b) 3 consecutive days of dipstick UA ≥ 3+ with the most recent urine sample in that 3-day period determined to have a UPCR > 0.2 g/g or (c) dipstick UA protein ≥ 3+ with edema on any day with a urine sample determined to have a UPCR > 0.2 g/g; or (2) any systemic corticosteroid use for more than 14 days within a 30-day period. In some embodiments, the subject's complete remission is maintained through treatment week 52 without initiation of any rescue therapy for INS other than systemic corticosteroids.

In some embodiments, the methods of the present disclosure produce a depletion of circulating peripheral B cells in an individual. In some embodiments, the circulating peripheral B cells are CD19+ B cells. In some embodiments, the circulating peripheral B cells are naïve B cells. In some embodiments, the circulating peripheral B cells are memory B cells. In some embodiments, the circulating peripheral B cells are plasmablasts or plasma cells. In some embodiments, after administration of a type II anti-CD20 antibody of the present disclosure ( e.g. , according to any of the methods described herein), the level of circulating peripheral B cells in peripheral blood is about 7 cells per µL or less, about 6 cells per µL or less, about 5 cells per µL or less, about 4 cells per µL or less, about 3 cells per µL or less, about 2 cells per µL or less, about 1 cell per µL or less, or about 0.5 cells per µL or less. In some embodiments, the level of circulating peripheral B cells is measured using high sensitivity flow cytometry (HSFC) as described herein. In some embodiments, B cells are depleted to a level below the limit of detection using HSFC. In some embodiments, the HSFC has a lower limit of quantification (LLOQ) for B cells of about 1.0 cells per µL or less, about 0.8 cells per µL or less, about 0.6 cells per µL or less, about 0.5 cells per µL or less, or about 0.441 cells per µL or less. In some embodiments, circulating peripheral B cells in a subject are depleted by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%. In some embodiments, circulating peripheral B cell depletion persists for at least 52 weeks after the first dose of the first antibody exposure. In some embodiments, circulating peripheral B cell depletion persists for at least 51 weeks, at least 50 weeks, at least 49 weeks, at least 48 weeks, at least 47 weeks, at least 46 weeks, at least 45 weeks, at least 44 weeks, at least 43 weeks, at least 42 weeks, at least 41 weeks, at least 40 weeks, at least 39 weeks, at least 38 weeks, at least 37 weeks, at least 36 weeks, at least 35 weeks, at least 34 weeks, at least 33 weeks, at least 32 weeks, at least 31 weeks, at least 30 weeks, at least 29 weeks, at least 28 weeks, at least 27 weeks, at least 26 weeks, at least 28 weeks, at least 29 ... In some embodiments, circulating peripheral B cell depletion refers to circulating peripheral B cell depletion obtained after a first antibody exposure ( e.g. , including 1 or 2 doses of an anti-CD20 antibody as described herein), after a second antibody exposure ( e.g. , including 1 or 2 doses of an anti-CD20 antibody as described herein), 3 months after treatment ( e.g. , after receiving a first and/or second antibody exposure as described herein), 6 months after treatment ( e.g. , after receiving a first and/or second antibody exposure as described herein), 9 months after treatment ( e.g. , after receiving a first and/or second antibody exposure as described herein), 12 months after treatment ( e.g. , after receiving a first and/or second antibody exposure as described herein). Cell measurements, for example , compared to corresponding measurements in the same individual before treatment, or compared to corresponding measurements in a control individual ( e.g. , an individual who has not received treatment).

Methods for analyzing circulating peripheral B cell depletion in an individual are known in the art, for example , flow cytometry using one or more antibodies that recognize B cell markers. In some embodiments, high sensitivity flow cytometry (HSFC) can be used to determine circulating peripheral B cell depletion (see , e.g., Vital, EM et al. , (2011) Arthritis Rheum. 63:3038-3047 and Example 1). In some embodiments, the B cells are CD19+ B cells. In some embodiments, the B cells are naïve B cells ( e.g., CD19+CD27- B cells), memory B cells ( e.g., CD19+CD27+ B cells), or plasmablasts ( e.g., CD19+CD27+CD38++ B cells). In some embodiments, the B cells are CD19+CD3-CD14- cells and/or CD19+CD33-CD56- cells. In some embodiments, the B cells are CD19+CD3-CD14-CD33-CD56- cells. In some embodiments, the B cells include CD19+CD20+ B cells, CD19+CD20- B cells, and CD19+CD22+ B cells. In some embodiments, the B cells are circulating peripheral B cells, such as from a peripheral blood sample.

In some embodiments, the level of circulating peripheral B cells present in a peripheral blood sample is measured ( e.g., by HSFC) as follows. Lymphocytes in the sample are identified by flow cytometry ( e.g. , by plotting a CD45 versus side scatter dot plot and circling CD45+ cells). In some embodiments, doublets are excluded from analysis prior to this step ( e.g. , by circling single cells and excluding forward scatter and/or side scatter doublets). CD19+ B cells are then identified by excluding T cells, NK cells, and monocytes. For example, CD19+CD3-CD14- cells can be identified from parental CD45+ lymphocytes ( e.g. , by plotting CD19 versus CD3/CD14 and gated on CD19+CD3-CD14- cells), and CD19+CD33-CD56- B cells can be identified from parental CD19+CD3-CD14- cells ( e.g. , by plotting CD19 versus CD33/CD56 and gated on CD19+CD33-CD56- cells). The B cell count can then be determined , for example, by dividing the number of CD19+ B cells detected ( e.g., CD19+CD3-CD14-CD33-CD56- cells) by the sample volume. In some embodiments, the number of beads or other QC controls is also quantified and the B cell count can then be determined, for example, by calculating (CD19+ events x bead count)/(number of beads x sample volume).

In some embodiments, after administration of a type II anti-CD20 antibody of the present disclosure ( e.g. , according to any of the methods described herein), circulating peripheral B cells in peripheral blood are about 7 cells per µL or less, about 6 cells per µL or less, about 5 cells per µL or less, about 4 cells per µL or less, about 3 cells per µL or less, about 2 cells per µL or less, about 1 cell per µL or less, or about 0.5 cells per µL or less, such as 5 cells per µL or less. In some embodiments, B cells are depleted to a level below the detectable limit using HSFC. In some embodiments, the HSFC has a lower limit of quantification (LLOQ) of about 1.0 cells per µL or less, about 0.8 cells per µL or less, about 0.6 cells per µL or less, about 0.5 cells per µL or less, or about 0.441 cells per µL or less for B cells. IV. Articles or Kits

In another aspect, an article of manufacture or kit comprising a type II anti-CD20 antibody of the present disclosure that can be used in any of the methods described herein (e.g., for the treatment, prevention and/or diagnosis of a disease described herein) is provided. The article of manufacture or kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container can hold a composition that is used by itself or in combination with another composition that is effective for treating, preventing and/or diagnosing a disease or for depleting circulating peripheral B cells, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle). At least one active agent in the composition is an antibody described herein ( e.g. , a type II anti-CD20 antibody disclosed herein). The label or package insert indicates that the composition is used to treat a selected condition or depletion of circulating peripheral B cells according to any of the methods described herein. Alternatively or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. From a commercial and user perspective, it may further comprise other materials, including other buffers, diluents, filters, needles, and syringes.

In some embodiments, provided herein is a product or kit comprising: a container comprising a type II anti-CD20 antibody of the present disclosure and, optionally, a pharmaceutically acceptable carrier, and, optionally, a package insert comprising instructions for treating childhood-onset INS in an individual or reducing the risk and/or frequency of recurrence of childhood-onset INS, for example , wherein the instructions for use instruct that a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody be administered to the individual until about 18 weeks to about 26 weeks after the first exposure, before providing the second antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises between about 1800 mg and about 2200 mg. wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg. In some embodiments, the instructions for use indicate that the individual is greater than or equal to 2 years old and less than 25 years old. In some embodiments, the instructions indicate that the individual weighs greater than or equal to 45 kg. In some embodiments, the antibody is obinutuzumab.

In some embodiments, provided herein is a product or kit comprising: a container comprising a Type II anti-CD20 antibody of the present disclosure and, optionally, a pharmaceutically acceptable carrier, and, optionally, a package insert comprising instructions for treating childhood-onset INS in an individual or reducing the risk and/or frequency of recurrence of childhood-onset INS, for example , wherein the instructions for use instruct to administer to the individual a first antibody exposure to a Type II anti-CD20 antibody and a second antibody exposure to the Type II anti-CD20 antibody until about 18 weeks to about 26 weeks after the first exposure, before providing the second antibody exposure; wherein the first antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the first antibody exposure comprises a Type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg; wherein the second antibody exposure comprises one or two doses of a type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of the type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg. In some embodiments, the instructions for use indicate that the individual is greater than or equal to 2 years old and less than 25 years old. In some embodiments, the instructions for use indicate that the individual weighs less than 45 kg. In some embodiments, the antibody is obinutuzumab.

The article of manufacture or kit may further comprise a second or third container comprising a second drug, wherein the anti-CD20 antibody ( e.g., type II anti-CD20 antibody of the present disclosure) is the first drug, wherein the article further comprises instructions for use on the package insert for treating the subject with the second drug. The article of manufacture in these embodiments of the present invention may further comprise package instructions indicating that the composition can be used to treat a specific disease.

This specification is considered sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention other than those shown and described herein are apparent to one skilled in the art from the foregoing description and are within the scope of the accompanying patent applications. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. Examples

The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention. It should be understood that the examples and embodiments as described herein are for illustrative purposes only, and various modifications or variations based on the described examples and embodiments will be suggested to those skilled in the art and will be included within the spirit and scope of this application and the scope of the attached patent applications. Example 1 : A Phase III , multicenter, randomized, open-label study to evaluate the efficacy and safety of obinutuzumab and MMF in patients with childhood-onset idiopathic nephrotic syndrome

Presented below is a Phase III, randomized, open-label, multicenter, active comparative study designed to evaluate the efficacy, safety, and pharmacokinetics (PK)/pharmacodynamics (PD) of obinutuzumab versus MMF in maintaining remission in participants with childhood-onset frequently relapsing nephrotic syndrome (FRNS) or steroid-dependent nephrotic syndrome (SDNS) who were in complete remission at study entry and were considered to be at high risk for relapse.

Patients with childhood-onset FRNS/SDNS are at risk for short- and long-term steroid toxicity, serious infections, edema, thromboembolic events, and acute renal injury during relapses. Reduction of these risks depends on identification of effective therapies and maintenance of clinical remission via sustained reductions in proteinuria. The current standard of care (SOC) remains limited to the combination of systemic corticosteroids and immunosuppressants, but most available regimens produce complete renal response and remission in less than half of treated patients. Goals and Endpoints

This study evaluates the efficacy, safety, pharmacokinetics, and pharmacodynamics of obinutuzumab and MMF in participants aged ≥ 2 years and ≤ 25 years with childhood-onset FRNS or SDNS. The specific primary and secondary objectives of the study and the corresponding endpoints are summarized below. In this protocol, "study treatment" refers to the treatment assigned to a participant as part of the study ( i.e. , obinutuzumab or MMF).

The primary objective of the study was to evaluate the efficacy of obinutuzumab compared with MMF in participants aged ≥ 2 years and ≤ 25 years with childhood-onset FRNS or SDNS. The primary endpoint was the proportion of participants who achieved sustained complete remission at 1 year, defined as first morning fasting urine protein-to-creatinine ratio (UPCR) ≤ 0.2 g/g at Week 52, without relapse or any of the following complications. Concurrent events occurring after Week 8 included: (1) relapse, as defined by any of the following events: (a) first morning fasting UPCR ≥ 2 g/g, (b) 3 consecutive days of dipstick urinalysis (UA) ≥ 3+ (home monitoring) with the most recent urine sample in that 3-day period determined to have a UPCR > 0.2 g/g as measured by a central laboratory, or (c) dipstick UA protein ≥ 3+ with edema on any day with a urine sample determined to have a UPCR > 0.2 g/g; and (2) any systemic corticosteroid use for more than 14 days within a 30-day period. Complications occurring after randomization included: (1) initiation of any rescue therapy for INS other than systemic corticosteroids, as determined by the investigator's best medical judgment; (2) discontinuation of treatment due to lack of efficacy; and/or (3) death.

The secondary objective was to evaluate the efficacy of obinutuzumab compared with MME. Corresponding secondary endpoints were: (1) overall relapse-free survival (RFS); (2) probability of RFS at week 52; (3) cumulative corticosteroid dose; (4) number of relapses during the randomized study treatment period; (5) proportion of participants who experienced relapse-related edema during the 52-week treatment period; and (6) proportion of patients with durable complete remission at week 76.

Another secondary objective was to assess changes in fatigue in participants treated with obinutuzumab compared with those treated with MMF. The corresponding secondary endpoint was the mean change from baseline to week 52 in the “general fatigue” domain of the Pediatric Quality of Life Inventory (PedsQL)-Multidimensional Fatigue Inventory total score.

The third secondary objective was to assess changes in quality of life in participants treated with obinutuzumab compared with those treated with MMF. The corresponding secondary endpoint was the mean change from baseline to week 52 in the "physical functioning" domain of the PedsQL Quality of Life Inventory.

The fourth secondary objective was to assess edema over time. The corresponding secondary endpoint was the mean change from baseline to week 52 on the Cure Glomerulonephropathy (CureGN) Edema Scale.

The fifth secondary objective was to evaluate the safety of obinutuzumab compared with MMF. The corresponding secondary endpoints were: (1) the incidence, nature, and severity of adverse events (AEs), where severity was determined by AE intensity (mild, moderate, severe, life-threatening) and National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) grade, from baseline to Week 52, and (2) the incidence of laboratory or vital sign abnormalities from baseline to Week 52.

The sixth secondary objective was to characterize the PK profile of obinutuzumab. The corresponding secondary endpoint was the serum concentration of obinutuzumab at the specified time points.

The seventh secondary objective was to characterize obinutuzumab-induced changes in PD. The corresponding secondary endpoints were: (1) the proportion of participants who achieved B cell depletion ( e.g. , using high-sensitivity flow cytometry (HSFC)) at designated time points; and (2) total peripheral B cell and B cell subset ( e.g. , memory B cell) counts and changes from baseline at designated time points.

The exploratory objective was to evaluate the efficacy of obinutuzumab compared with MMF. Corresponding exploratory endpoints were: (1) change in UPCR from baseline to Week 52; (2) change in estimated glomerular filtration rate (eGFR) from baseline to Week 52; (3) proportion of participants achieving sustained peripheral B-cell depletion at Week 24, Week 52, and Week 76; (4) proportion of participants achieving RFS at Week 24; (5) proportion of participants in sustained complete remission at Week 76; (6) change in physician's global assessment of disease activity (PGA) from baseline to Week 24 and Week 52; and (7) change in individual's global assessment of disease activity (SGA) from baseline to Week 76. Changes at Weeks 24 and 52; and (8) the proportion of participants who remained relapse-free before completing steroid taper.

The second exploratory objective was to explore the relationship between exposure efficacy and exposure safety. The corresponding exploratory endpoints were: (1) changes in obinutuzumab exposure and selected efficacy endpoints (including durable complete remission (SCR) and RFS) from baseline to Week 52 and over time; and (2) changes in obinutuzumab exposure and the incidence, nature, and severity of selected adverse events from baseline to Week 52 and over time.

The third exploratory objective was to evaluate the potential relationship between drug exposure and B cell depletion. The corresponding exploratory endpoints were: (1) changes in obinutuzumab exposure and circulating CD19+ B cell numbers relative to baseline over time; and (2) total peripheral B cell and B cell subset ( e.g. , memory B cell) counts before/at relapse.

The fourth exploratory objective was to assess the immune response to obinutuzumab. The corresponding exploratory endpoints were the proportion of participants with anti-drug antibodies (ADA) at baseline and the incidence of ADA after treatment during the study.

The fifth exploratory objective was to evaluate the potential effect of ADA. The corresponding exploratory endpoints were the relationship between ADA status and efficacy, safety, PD, or PK endpoints.

The sixth exploratory objective is to identify and/or evaluate biomarkers that provide evidence of obinutuzumab activity ( i.e. , efficacy biomarkers) or increase knowledge and understanding of disease biology and drug safety. The corresponding exploratory endpoints are the relationship between biomarkers in blood and efficacy, safety, PK, immunogenicity or other biomarker endpoints. Inclusion and Exclusion Criteria

Inclusion criteria for the study included: (1) participants aged ≥ 2 years and ≤ 25 years at the time of randomization; (2) diagnosed with FRNS or SDNS before age 18 years according to international guidelines [ e.g. , KDIGO 2021, IPNA 2023 ( see Trautmann et al. (2023) Pediatr Nephrol 38:877-919]; (3) in complete remission defined by the absence of edema, UPCR ≤ 0.2 g/g at screening, and trace or negative urine dipstick readings three times daily in the week before randomization; (4) 6 months before screening. (5) patients who had received cyclophosphamide within 6 months before randomization must have experienced at least 1 relapse after cyclophosphamide cessation; and (6) estimated glomerular filtration rate (eGFR) within the normal range for age (using the modified Schwartz formula if younger than 18 years of age or the Chronic Kidney Disease Epidemiology Institute (CKD-EPI) formula if 18 years of age or older). Frequently relapsing nephrotic syndrome (FRNS) was defined as ≥ 2 relapses every 6 months within 6 months of disease onset or ≥ 4 relapses every 12 months within the next 12 months. Steroid-dependent nephrotic syndrome (SDNS) was defined as 2 consecutive relapses during treatment with prednisone or penicillin (at full dose or during tapering) or within 15 days after cessation of prednisone or penicillin.

Exclusion criteria included: (1) secondary nephrotic syndrome ( i.e. , reflux nephropathy, IgA nephropathy, lupus nephritis , etc. ); (2) history of steroid-resistant nephrotic syndrome; (3) history of a known genetic defect that directly causes nephrotic syndrome ( i.e. , NPHS2 [podocin], NPHS1 [nephrin], PLCE1, WT1, or other known genetic causes); (4) treatment with immunosuppressive medications other than MMF or oral corticosteroids to prevent relapse within 2 months before randomization; (5) history of organ or bone marrow transplantation; (6) participation in another therapeutic trial within 30 days of enrollment or within 5 half-lives of the study drug (whichever is longer); (7) Intolerance or contraindication to study therapy, including any of the following: (a) severe allergy or history of anaphylaxis to monoclonal antibodies or known allergy to any component of the obinutuzumab infusion, (b) lack of peripheral venous access, (c) intolerance or contraindication to oral or IV corticosteroids, or (d) intolerance or contraindication to MMF; (8) patients who have demonstrated failure of prior MMF therapy, as defined by two or more relapses in any 6-month period while receiving MMF for at least 6 months of duration; (9) participants who, in the investigator's judgment, may require systemic corticosteroids during the study for reasons other than idiopathic nephrotic syndrome; (10) receiving excluded therapies (see below); (11) Active infection of any kind (except nail bed fungal infection) or any major infectious episode requiring hospitalization or treatment with IV antibiotics within 4 weeks prior to screening, or completion of oral antibiotics within 2 weeks prior to randomization; (12) Evidence of active tuberculosis (TB) infection; (13) History of or current active primary or secondary immunodeficiency, including known history of HIV infection and other severe immunodeficiency blood diseases; (14) History of severe recurrent or chronic infection; (15) History of progressive multifocal leukoencephalopathy; (15) Past 5 History of cancer within the past year or current cancer, including solid tumors, hematologic malignancies, and carcinoma in situ (excluding resected and treated basal cell carcinoma and squamous cell carcinoma of the skin); (16) Major surgery requiring hospitalization within 4 weeks prior to screening or during the screening period; (17) High risk for clinically severe bleeding or any condition requiring plasma exchange, intravenous immunoglobulin, or acute blood product transfusion; (18) Evidence of any significant or uncontrolled concomitant disease that, in the judgment of the investigator, would preclude the participant's participation, including but not limited to nervous system, respiratory system, cardiac, hepatic, endocrine, malignant, or gastrointestinal diseases; (19) Current or history of alcohol or drug abuse; and (20) any of the following laboratory parameters at screening: —AST or ALT > 2.5 × upper limit of normal (ULN) (for age and sex) not attributable to an underlying nephrotic syndrome —Amylase or lipase > 2 x ULN —Absolute neutrophil count < 1.5 × 10 ³ /µL —Hemoglobin < 8 g/dL —Platelet count < 110,000/µL for participants 12 years of age or younger; platelet count < 50,000/µL for participants 12 years of age or older —Hepatitis B surface antigen positive— B Hepatitis C antibody positive - Hepatitis B virus core antibody positive - Serum human chorionic gonadotropin positive at screening Excluded treatments include: - Use of cyclophosphamide, levamisole, imidazolidinedione, tacrolimus, cyclosporine, or voclosporin during the 2 months before screening or during the screening period. - Any biologic B-cell depleting therapy ( e.g. , anti-CD19, anti-CD20, anti-CD22), such as but not limited to rituximab, ocrelizumab, or ofatumumab, within 9 months prior to the Day 1 baseline visit - Any biologic therapy (other than anti-CD19, anti-CD20, anti-CD22), such as but not limited to belimumab, daratumumab, ustekinumab, anifrolumab, secukinumab, or atacicept, during the 2 months prior to screening or during the screening period - Receipt of Janus cytokine during the 2 months prior to screening or during the screening period Oral inhibitors of JAK-related kinases (JAK), BTK, or TYK2, including baricitinib, tofacitinib, upadacitinib, filgotinib, ibrutinib, or fenebrutinib, or any investigational agent—received any live vaccine investigational treatment during the 28 days prior to screening or during screening

The study consisted of four phases: a screening period of up to 28 days, an initial open-label treatment period of 52 weeks, an extension period after week 52, and a safety follow-up (SFU) period of a minimum of 12 months from the time of completion or cessation of study treatment. A schematic diagram of the study is provided in Figure 1 .

Approximately 80 participants aged ≥ 2-25 years were randomized in a 1:1 ratio to one of two open-label treatment groups: Arm A (obinutuzumab) or Arm B (MMF). Randomization was stratified by participant type of disease (FRNS vs. SDNS) and use of immunosuppressive therapy for INS other than corticosteroids before study entry (MMF/other immunosuppressants vs. no MMF/other immunosuppressants).

The investigational product used in this study was obinutuzumab.

After a 28-day (+/-7-day) screening period, randomized participants entered a 52-week treatment period. During the treatment period, participants received obinutuzumab 1000 mg IV infusion on Days 1 , 15, 168 (Week 24), and 182 (Week 26) of the initial 52-week treatment period, or daily oral MMF ( tablet , capsule, or liquid formulation) starting on Day 1 or continuing according to Figure 1. Participants weighing 45 kg or more received a 1000 mg dose of obinutuzumab. Participants weighing less than 45 kg received a weight-adjusted dose of 20 mg/kg for the obinutuzumab infusion. Methylphenidate is administered as a premedication prior to infusion at 80 mg IV (1.5 mg/kg if ≤45 kg). Acetaminophen/paraacetaminophen is administered as a premedication prior to infusion at 15 mg/kg (maximum dose 1000 mg) PO. Diphenhydramine is administered as a premedication prior to infusion at 0.5-1 mg/kg (maximum dose 50 mg) PO or IV.

Participants randomized to MMF took a target dose of 1200 mg/m2/ ^day (maximum 2.5 g/day) in divided doses. Participants receiving daily oral prednisone (or penicillin equivalent) at randomization tapered to a target of 0 mg/day by Week 8 after randomization (or earlier, e.g. , Study Weeks 4-6, if applicable) and continued without prednisone for the remainder of the study. Participants who experienced a disease relapse received prednisone or penicillin up to 2 mg/kg/day (60 mg/ ^m2 /day, maximum dose 60 mg/day) until urine protein dipstick was negative/trace (or UPCR ≤ 0.2 g/g) for 3 or more consecutive days, followed by a 4-week taper of oral corticosteroids while continuing study treatment. If participants in the MMF group met criteria for rescue therapy, patients were considered to meet the definition of a concurrent event and received obinutuzumab (2 × 1000 mg 14 days apart or 20 mg/kg if < 45 kg) or an alternative therapy for INS. If participants in the obinutuzumab group met criteria for rescue therapy, patients were considered to meet the definition of a complication and received daily MMF (600 mg/m ² BID [target 1200 mg/m ² ] in divided doses, maximum 2 g/day) or alternative therapy at the investigator's discretion to treat the relapse. MMF was discontinued while taking obinutuzumab.

The primary endpoint of the proportion of participants with sustained complete remission (UPCR ≤ 0.2 g/g without relapse or other complications) at 1 year was assessed when the last randomized participant completed Week 52.

After the Week 52 assessment, participants could continue on obinutuzumab in the treatment extension phase until the common end date (CCOD) or proceed directly to the safety follow-up (SFU). Participants who presented with a relapse after Week 52 were treated according to the investigator’s best judgment, which could include administration of the initial dose or additional doses of obinutuzumab, either in the MMF group or in the obinutuzumab group. Patients who did not meet the relapse criteria at or after Week 52 were not eligible to receive obinutuzumab in the extension phase but were followed every 3 months at study visits until the CCOD. Participants with peripheral B cell depletion were followed every 12 weeks for 6 months in the SFU and then every 6 months until peripheral CD19 B cells recovered to pre-treatment values or to the central laboratory normal range for this patient population, whichever was lower, until the end of the study.

The first SFU visit will be scheduled approximately 12 weeks after the last study visit of the treatment period or CCOD, whichever occurs first. No obinutuzumab infusions or MMF will be given during the SFU. Standard of care will be provided at the investigator's discretion. Patients who do not receive obinutuzumab will have only one SFU visit, with assessments specified at SFU Visit 1. Patients who receive obinutuzumab will be followed in the SFU until they meet both of the following criteria: (1) peripheral B cells have recovered to pre-obinutuzumab baseline levels or to the normal range for the population, whichever is lower; and (2) the last infusion of obinutuzumab was at least 12 months ago.

Absolute CD19+ B cell counts will be measured at each SFU visit. For participants with persistent B cell depletion (defined as an absolute CD19+ B cell count below the pre-treatment nadir and less than the lower limit of normal (LLN) for that population) who are not receiving additional therapy associated with a decrease in peripheral B cells, SFU visits will continue every 6 months until either: (1) peripheral CD19+ B cells recover to the pre-treatment nadir or the age-specific LLN for that patient population, whichever is lower; (2) additional therapy associated with a decrease in peripheral B cells (e.g., belimumab) is received. (belimumab, rituximab, or cyclophosphamide, or off-study use of obinutuzumab); or (3) end of study.

Participants complete the study when: (1) they complete the SFU requirement; (2) their last study visit on or after CCOD and the investigator intends to treat the participant for nephrotic syndrome outside the study protocol without completing the required SFU; or (3) the study ends. After study completion, eligible participants will be offered the opportunity to continue their Roche investigational medicinal product (IMP; obinutuzumab) by the study sponsor. Study Duration

The minimum duration of study participation for each individual is expected to be approximately 1.5-2 years (for all patients who have received obinutuzumab, the SFU will be a minimum of 12 months after the last obinutuzumab infusion). Participants may continue to participate in the study and be followed or receive repeat treatments based on their activity schedule until the last enrolled participant has been in the study for at least 18 months.

If peripheral B cells remain below LLN at clinical cutoff, the maximum duration of study participation is estimated to be approximately 3 years or longer. In this case, participants will be asked to return for SFU visits every 12 weeks for 6 months and then every 6 months until B cells recover to pre-obinutuzumab dose baseline or normal central laboratory LLN for the participant population, or until the end of the study.

Figure 1 provides a schematic diagram of a controlled study of childhood-onset INS ( e.g. , recurrent recurrent nephrotic syndrome or steroid-dependent nephrotic syndrome) in patients aged ≥ 2-25 years using the type II anti-CD20 antibody obinutuzumab. BID = twice daily; CCOD = common end date of primary analysis; FRNS = recurrent recurrent nephrotic syndrome; MMF = mycophenolate mofetil; PO = oral; SDNS = steroid-dependent nephrotic syndrome; SFU = safety follow-up; Wk = week. ^a Administration of the first dose of study treatment (Day 1) should occur within 24 hours after baseline assessment. However, administration for up to 72 hours was permitted if necessary. The second infusion should occur on Day 15 ± 1 day. ^bThe primary efficacy endpoint was the proportion of participants with a sustained complete remission at one year, measured at Week 52.

TW202434286A_112142876_SEQL.xml

Claims (50)

A method of treating childhood-onset idiopathic nephrotic syndrome (INS) in an individual, comprising administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; wherein the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, the first antibody exposure comprising: (a) a total exposure to the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure to the type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg, provided that the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the second antibody exposure comprises: (c) a total exposure to the Type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure to the Type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg, provided that the individual weighs less than 45 kg; wherein the Type II anti-CD20 antibody is obinutuzumab; and wherein the individual is a human who is greater than or equal to 2 years of age and less than or equal to 25 years of age. A method of reducing the risk and/or frequency of relapse in an individual with childhood-onset idiopathic nephrotic syndrome (INS), comprising administering to the individual a first antibody exposure to a Type II anti-CD20 antibody and a second antibody exposure to the Type II anti-CD20 antibody; wherein the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the first antibody exposure comprising: (a) a total exposure of between about 1800 mg and about 2200 mg of the Type II anti-CD20 antibody, or (b) between about 36 mg/kg and about 44 mg/kg of the Type II anti-CD20 antibody. total exposure to the Type II anti-CD20 antibody, provided that the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the second antibody exposure comprising: (c) a total exposure to the Type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure to the Type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg, provided that the individual weighs less than 45 kg; wherein the Type II anti-CD20 antibody is obinutuzumab; and wherein the individual is a human who is greater than or equal to 2 years of age and less than or equal to 25 years of age. The method of claim 1 or claim 2, wherein the first antibody exposure comprises a total exposure to the type II anti-CD20 antibody between about 1800 mg and about 2200 mg; wherein the second antibody exposure comprises a total exposure to the type II anti-CD20 antibody between about 1800 mg and about 2200 mg; and wherein the individual weighs greater than or equal to 45 kg. The method of any one of claims 1 to 3, wherein the first antibody exposure comprises a first dose of the Type II anti-CD20 antibody between about 900 mg and about 1100 mg, and a second dose of the Type II anti-CD20 antibody between about 900 mg and about 1100 mg. The method of any one of claims 1 to 3, wherein the first antibody exposure comprises a first dose of the Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and a second dose of the Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and wherein the individual weighs less than 45 kg. The method of any one of claims 1 to 5, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and wherein the second dose of the first antibody exposure is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the first antibody exposure. The method of claim 6, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and wherein the second dose of the first antibody exposure is not provided until about 2 weeks after the first dose of the first antibody exposure. The method of any one of claims 1 to 4, 6 and 7, wherein the first dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody. The method of any one of claims 1 to 4 and 6 to 8, wherein the second dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody. The method of any one of claims 1 to 3 and 5 to 7, wherein the first dose of the first antibody exposure is about 20 mg/kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. The method of any one of claims 1 to 3, 5 to 7 and 10, wherein the second dose of the first antibody exposure is about 20 mg/kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. The method of any one of claims 1 to 4 and 6 to 9, wherein the second antibody exposure comprises a first dose of the Type II anti-CD20 antibody between about 900 mg and about 1100 mg, and a second dose of the Type II anti-CD20 antibody between about 900 mg and about 1100 mg. The method of any one of claims 1 to 3, 5 to 7, 10 and 11, wherein the second antibody exposure comprises a first dose of the Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and a second dose of the Type II anti-CD20 antibody between about 18 mg/kg and about 22 mg/kg, and wherein the individual weighs less than 45 kg. The method of any one of claims 1 to 13, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and wherein the second dose of the second antibody exposure is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the second antibody exposure. The method of claim 14, wherein the second dose of the second antibody exposure is not provided until about 2 weeks after the first dose of the second antibody exposure. The method of any one of claims 1 to 4, 6 to 9, 12, 14 and 15, wherein the first dose of the second antibody exposure is about 1000 mg of the type II anti-CD20 antibody. The method of any one of claims 1 to 4, 6 to 9, 12 and 14 to 16, wherein the second dose of the second antibody exposure is about 1000 mg of the type II anti-CD20 antibody. The method of any one of claims 1 to 3, 5 to 7, 10, 11 and 13 to 15, wherein the first dose of the second antibody exposure is about 20 mg/kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. The method of any one of claims 1 to 3, 5 to 7, 10, 11, 13 to 15 and 18, wherein the second dose of the second antibody exposure is about 20 mg/kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. The method of any one of claims 1 to 19, wherein the individual has or has been diagnosed with childhood-onset INS. The method of any one of claims 1 to 20, wherein the childhood-onset INS is frequently recurrent nephrotic syndrome (FRNS). The method of any one of claims 1 to 20, wherein the childhood-onset INS is steroid-dependent nephrotic syndrome (SDNS). The method of any one of claims 1 to 22, wherein the subject is in complete remission prior to the administering. The method of any one of claims 1 to 23, further comprising administering to the individual an effective amount of a glucocorticoid or a corticosteroid. The method of claim 24, wherein the glucocorticoid or corticosteroid comprises methylprednisolone. The method of claim 25, wherein methylprazosin is administered intravenously to the subject in an amount of 80 mg. The method of claim 25, wherein methylprazosin is administered intravenously to the subject at a dose of 1.5 mg/kg; and wherein the subject weighs less than 45 kg. The method of claim 24, wherein the glucocorticoid or corticosteroid comprises prednisone. The method of any one of claims 1 to 28, further comprising administering to the individual an effective amount of an antihistamine. The method of claim 29, wherein the antihistamine comprises diphenhydramine. The method of claim 30, wherein diphenhydramine hydrochloride is administered orally or intravenously to the subject in an amount of 0.5 to 1 mg/kg. The method of any one of claims 1 to 31, further comprising administering to the individual an effective amount of acetaminophen. The method of claim 32, wherein the acetaminophen is administered orally at a dose of 15 mg/kg and a maximum dose of 1000 mg. The method of any one of claims 1 to 33, wherein the method induces sustained complete remission in the individual at 1 year. The method of claim 1 or claim 2, wherein the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on day 1 and day 15 of treatment; and wherein the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on day 168 and day 182 of treatment. The method of claim 1 or claim 2, wherein the first antibody exposure comprises two doses of 20 mg/kg of the type II anti-CD20 antibody on day 1 and day 15 of treatment; wherein the second antibody exposure comprises two doses of 20 mg/kg of the type II anti-CD20 antibody on day 168 and day 182 of treatment; and wherein the individual weighs less than 45 kg. The method of claim 1 or claim 2, wherein the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody at week 0 and week 2 of treatment; and wherein the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody at week 24 and week 26 of treatment. The method of claim 1 or claim 2, wherein the first antibody exposure comprises two doses of 20 mg/kg of the type II anti-CD20 antibody at week 0 and week 2 of treatment; wherein the second antibody exposure comprises two doses of 20 mg/kg of the type II anti-CD20 antibody at week 24 and week 26 of treatment; and wherein the individual weighs less than 45 kg. A method for treating childhood-onset INS in an individual or reducing the risk and/or frequency of recurrence thereof, comprising administering to the individual intravenously a first antibody exposure and a second antibody exposure to obinutuzumab; wherein the first antibody exposure comprises two doses of 1000 mg of obinutuzumab at week 0 and week 2 of treatment; wherein the second antibody exposure comprises two doses of 1000 mg of obinutuzumab at week 24 and week 26 of treatment; wherein the individual is a human who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the individual weighs greater than or equal to 45 kg. A method for treating childhood-onset INS in an individual or reducing the risk and/or frequency of recurrence thereof, comprising administering to the individual intravenously a first antibody exposure and a second antibody exposure to obinutuzumab; wherein the first antibody exposure comprises two doses of 20 mg/kg of obinutuzumab at week 0 and week 2 of treatment; wherein the second antibody exposure comprises two doses of 20 mg/kg of obinutuzumab at week 24 and week 26 of treatment; wherein the individual is a human who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the individual weighs less than 45 kg. A method of treating childhood-onset INS in an individual or reducing the risk and/or frequency of recurrence thereof, comprising administering to the individual intravenously a first antibody exposure and a second antibody exposure to obinutuzumab; wherein the first antibody exposure comprises two doses of 1000 mg of obinutuzumab on day 1 and day 15 of treatment; wherein the second antibody exposure comprises two doses of 1000 mg of obinutuzumab on day 168 and day 182 of treatment; wherein the individual is a human who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the individual weighs greater than or equal to 45 kg. A method for treating childhood-onset INS in an individual or reducing the risk and/or frequency of recurrence thereof, comprising administering to the individual intravenously a first antibody exposure and a second antibody exposure to obinutuzumab; wherein the first antibody exposure comprises two doses of 20 mg/kg of obinutuzumab on day 1 and day 15 of treatment; wherein the second antibody exposure comprises two doses of 20 mg/kg of obinutuzumab on day 168 and day 182 of treatment; wherein the individual is a human who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the individual weighs less than 45 kg. The method of claim 39 or claim 40, further comprising administering methylprednisolone by intravenous (IV) infusion to the individual at week 0, week 2, week 24, and week 26 of treatment prior to administering obinutuzumab. The method of claim 41 or claim 42, further comprising administering methylprednisolone by intravenous (IV) infusion to the individual on day 1, day 15, day 168, and day 182 of treatment prior to administering obinutuzumab. The method of claim 43 or claim 44, wherein: (a) if the individual weighs 45 kg or more, 80 mg of methylprednisolone is administered to the individual; or (b) if the individual weighs less than 45 kg, 1.5 mg/kg of methylprednisolone is administered to the individual. A kit for treating childhood-onset INS in an individual, comprising: (a) a container comprising a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody is obinutuzumab; (b) a package insert having instructions for treating childhood-onset INS in an individual, wherein the instructions indicate that the individual is a human aged greater than or equal to 2 years and less than or equal to 25 years; and wherein the instructions further indicate that a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody are administered to the individual until about 18 weeks to about 26 weeks after the first antibody exposure, before providing the second antibody exposure; wherein the first antibody exposure comprises the type II anti-CD20 antibody; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, the first antibody exposure comprising a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, the second antibody exposure comprising a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg. A kit for treating childhood-onset INS in an individual, comprising: (a) a container comprising a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody is obinutuzumab; (b) a package insert having instructions for treating childhood-onset INS in an individual, wherein the instructions indicate that the individual is a human who is greater than or equal to 2 years old and less than or equal to 25 years old and weighs less than 45 kg; and wherein the instructions further indicate that a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody are administered to the individual such that the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, the first antibody exposure comprising a total exposure of the type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, the second antibody exposure comprising a total exposure of the type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg. A type II anti-CD20 antibody for use in a method of treating childhood-onset INS in a subject in need thereof, wherein the method comprises administering to the subject a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; wherein the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, the first antibody exposure comprising: (a) a total exposure of between about 1800 mg and about 2200 mg of the type II anti-CD20 antibody, or (b) between about 36 mg/kg and about 44 mg/kg of the type II anti-CD20 antibody. total exposure to the Type II anti-CD20 antibody, provided that the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the second antibody exposure comprising: (c) a total exposure to the Type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure to the Type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg, provided that the individual weighs less than 45 kg; wherein the Type II anti-CD20 antibody is obinutuzumab; and wherein the individual is a human who is greater than or equal to 2 years of age and less than or equal to 25 years of age. A type II anti-CD20 antibody for use in a method of reducing the risk and/or frequency of relapse in an individual with childhood-onset INS in need thereof, wherein the method comprises administering to the individual a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; wherein the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, the first antibody exposure comprising: (a) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure of the type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg total exposure to the Type II anti-CD20 antibody, provided that the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the Type II anti-CD20 antibody, the second antibody exposure comprising: (c) a total exposure to the Type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure to the Type II anti-CD20 antibody between about 36 mg/kg and about 44 mg/kg, provided that the individual weighs less than 45 kg; wherein the Type II anti-CD20 antibody is obinutuzumab; and wherein the individual is a human who is greater than or equal to 2 years of age and less than or equal to 25 years of age. A type II anti-CD20 antibody for use in the method of any one of claims 1 to 45.